Synthetic mimics of mir-34

ABSTRACT

Embodiments concern methods and compositions involving miR-34 mimics, including miR-34a and miR-34c mimics. In some embodiments, there are double-stranded RNA molecules with modified nucleotides having an active strand with a miR-34a sequence and a complementary passenger strand. In additional embodiments, there are double-stranded RNA molecules with modified nucleotides having an active strand with a miR-34c sequence and a complementary passenger strand.

This application claims priority to U.S. Provisional Patent Application61/439,280, which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates to the fields of molecular biology andmedicine. More specifically, there are methods and compositionsinvolving RNA molecules with at least the functional properties ofmiR-34 and in some embodiments, enhanced characteristics related tomiR-34 for the treatment of diseases and/or conditions. In someembodiments, RNA molecules that function as miR-34a or as miR-34c areprovided in these contexts.

II. Background

In 2001, several groups used a cloning method to isolate and identify alarge group of “microRNAs” (miRNAs) from C. elegans, Drosophila, andhumans (Lau et al., 2001; Lee and Ambros, 2001; Lagos-Quintana et al.,2003).

Published human mature microRNA sequences, described in the databasemiRBase 15.0 (Griffths-Jones et al., 2006), range in size from 16-27nucleotides in length and arise from longer precursors. The precursorsform structures that fold back on themselves in self-complementaryregions and are processed by the nuclease Dicer (in animals) or DCL1 (inplants) to generate the short double-stranded mature miRNA. One of themature miRNA strands is incorporated into a complex of proteins andmiRNA called the RNA-induced silencing complex (RISC). The miRNA guidesthe RISC complex to a target mRNA, which is then cleaved ortranslationally silenced, depending on the degree of sequencecomplementarity of the miRNA to its target mRNA. Currently, it isbelieved that perfect or nearly perfect complementarity leads to mRNAdegradation, as is most commonly observed in plants. In contrast,imperfect base pairing, as is primarily found in animals, leads totranslational silencing. However, recent data suggest additionalcomplexity (Bagga et al., 2005; Lim et al., 2005), and mechanisms ofgene silencing by miRNAs remain under intense study.

Studies have shown that changes in the expression levels of numerousmiRNAs are associated with various cancers (reviewed in Calin and Croce,2006; Esquela-Kerscher and Slack, 2006; Wiemer, 2007). miRNAs have alsobeen implicated in regulating cell growth and cell and tissuedifferentiation—cellular processes that associated with the developmentof cancer.

The activity of a variety of miRNAs has been identified and analyzed.Although effective miRNA mimics have been identified previously in U.S.Patent Application Publication 20080050744, which is hereby incorporatedby reference, there is a need for additional miRNA mimics that greatlyimprove one or more properties of the naturally occurring miRNA,particularly as these molecules move from the laboratory to the clinic.

SUMMARY OF THE INVENTION

Therapeutic microRNAs should be stable, active, and specificallyhybridize with the correct mRNA target. Embodiments concern miR-34a andmiR-34c mimics that have maintained and/or enhanced resistance tonuclease digestion, hybridization capability with the correct targetmRNAs, and/or functionality.

Embodiments concern different RNA molecules containing the sequence of amature miR-34a. RNA molecules may be double-stranded and/or blunt-ended,which means the molecule is double-stranded throughout the moleculeand/or blunt-ended on both ends. Moreover, embodiments concern chemicalmodifications of such RNA molecules to yield miR-34a mimics or miR-34cmimics with improved or enhanced properties. The active strand of adouble stranded RNA molecule contains the mature miR-34a sequence. Incertain embodiments, the sequence of one strand of a double stranded RNAmolecule consists of the sequence of a mature miR-34a sequence. In otherembodiments, the sequence of one strand of a double stranded RNAmolecule consists of the sequence of a mature miR-34c sequence.

In some embodiments there is an RNA molecule that is double-stranded,meaning the molecule is composed of two polynucleotides or strands thatcan be separated from one another. A double-stranded molecule does notinclude a hairpin molecule, which is one strand or polynucleotide. Insome embodiments, the RNA molecule is blunt-ended on one or both ends.In a double-stranded RNA molecule, one or both strands may be 18, 19,20, 21, 22, 23, 24, or 25 nucleotides in length, or any range derivabletherein. In certain embodiments, a double-stranded, blunt-ended moleculeis 22 or 23 basepairs (bps) in length.

It is contemplated that in some embodiments a double-stranded RNAmolecule contains two strands that are fully complementary to oneanother, which results in a molecule that is necessarily blunt-ended.

In certain embodiments, an RNA molecule has an active strand comprisinga mature human miR-34a sequence (5′-UGGCAGUGUCUUAGCUGGUUGU-3′) (SEQ IDNO:1) (22-mer). In certain embodiments, the mature miR-34a sequence hasthe sequence of SEQ ID NO:1 and an additional U at the 3′ end(5′-UGGCAGUGUCUUAGCUGGUUGUU-3′) (SEQ ID NO:2) (23-mer). Thus, in certainembodiments, an RNA molecule has an active strand with the sequence ofnucleotides 1 through 22 of SEQ ID NO:2. It is further contemplated thatin some embodiments, there is an RNA molecule having an active strandwith the sequence of nucleotides 1 through 22 of SEQ ID NO:2, whereinthe RNA molecule is 23 nucleotides in length because there is an A, C,G, or U at the 3′ end of the sequence. In some embodiments, the activestrand has a modified nucleotide at one or more internal positions.

In certain embodiments, an RNA molecule has an active strand comprisinga mature human miR-34c sequence (5′-AGGCAGUGUAGUUAGCUGAUUGC-3′) (SEQ IDNO:5) (23-mer). In some embodiments, the active strand has a modifiednucleotide at one or more internal positions.

By convention, sequences discussed herein are set forth 5′ to 3′ unlessother specified. Moreover, a strand containing the sequence of a SEQ IDNO has that sequence from 5′ to 3′ unless otherwise specified.

The term “internal positions” refers to a position that is neither thefirst nor last position in the strand. The term “modified nucleotide”means a nucleotide or nucleoside (if referring to the nucleobase at the5′ position) with an additional moiety or a replacement moiety comparedto an unmodified nucleotide. With active strands containing one or moremodified nucleotides, it is contemplated that there are, there are nofewer than, or there are no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, or 15 modified nucleotides, or any range derivable therein.It is specifically contemplated that in some embodiments, fewer thanevery nucleotide in the active strand is modified, and that fewer thanhalf of the nucleotides in the active strand are modified in certainembodiments. Moreover, in some embodiments, it is specificallycontemplated that an active strand having multiple modified nucleotidesdoes not have every nucleotide or every nucleotide in the active strandmodified. The miRNA mimics disclosed herein are sequence- and/orposition-specific.

In some embodiments methods concern a miR-34a mimic; such mimics includeRNA molecules having an active strand with a sequence that is identicalor that has 90% or more identity to SEQ ID NO:1 or SEQ ID NO:2. In otherembodiments, methods concern a miR-34c mimic; such mimics include RNAmolecules having an active strand with a sequence that is identical orthat has 90% or more identity to SEQ ID NO:5. In further embodiments,methods concern miRNA mimics that provide miR-34a and miR-34c activity;such mimics include RNA molecules having an active strand with asequence that is identical or that has 90% or more identity to SEQ IDNO:7.

In some embodiments, there is a double-stranded, blunt-ended RNAmolecule 22 or 23 basepairs in length comprising: a) an active strandcomprising SEQ ID NO:1 from 5′ to 3′ and b) a fully complementarypassenger strand comprising i) modified nucleotides in the first andlast two nucleotides of the passenger strand; and ii) a terminalmodification of the nucleotide at the 5′ end. In certain embodiments,this RNA molecule is 22 basepairs in length, while in other embodiments,this RNA molecule is 23 basepairs in length. In the latter case, theactive strand comprises SEQ ID NO:1, but the entire sequence is thesequence from SEQ ID NO:2. In additional embodiments, there is adouble-stranded, blunt-ended RNA molecule 23 or 24 basepairs in lengthcomprising: a) an active strand comprising SEQ ID NO:5 from 5′ to 3′ andat least one modified internal nucleotide and b) a fully complementarypassenger strand comprising i) modified nucleotides in the first andlast two nucleotides of the passenger strand; and ii) a terminalmodification of the nucleotide at the 5′ end. In additional embodiments,it is further contemplated that this RNA molecule comprises SEQ ID NO:1,SEQ ID NO:2, or SEQ ID NO:7 instead of SEQ ID NO:5. In some embodiments,the active strand comprises at least two modified nucleotides. Inadditional embodiments, the active strand does not have a modifiednucleotide in the first two positions at either end. In furtherembodiments, the active strand does not comprise a modified nucleotidein the first four positions from the 5′ end. In further embodiments, anRNA molecule has an active strand that comprises at least two modifiednucleotides, though the modified nucleotides are not in the first twopositions at the 5′ end of the active strand. In certain embodiments,the active strand does not comprise a modified nucleotide in the lasttwo positions of the active strand, that is, the last two positions atthe 3′ end.

In certain embodiments, an active strand with the sequence of SEQ IDNO:1 or SEQ ID NO:2 does not have any modified nucleotides, though insuch embodiments the passenger strand has at least a terminalmodification and at least one other modification. In additionalembodiments, an active strand with at least 90% identity to SEQ ID NO:1or SEQ ID NO:2 does not have any modified nucleotides, though in suchembodiments the passenger strand has at least a terminal modificationand at least one other modification. In even further embodiments, anactive strand with the sequence of SEQ ID NO:5 or SEQ ID NO:7 does nothave any modified nucleotides, though in such embodiments the passengerstrand has at least a terminal modification and at least one othermodification. In additional embodiments, an active strand with at least90% identity to SEQ ID NO:5 or SEQ ID NO:7 does not have any modifiednucleotides, though in such embodiments the passenger strand has atleast a terminal modification and at least one other modification.

In some embodiments, an active strand may comprise the mature miR-34asequence of SEQ ID NO:1 (5′-UGGCAGUGUCUUAGCUGGUUGU-3′) or comprise thesequence of nucleotides 1 through 22 of SEQ ID NO:2(5′-UGGCAGUGUCUUAGCUGGUUGUU-3′). SEQ ID NO:2 has the mature miR-34asequence of SEQ ID NO:1 in conjunction with an additional U at the 3′end. SEQ ID NO:2 has an extra U at the 3′ end compared to the maturehuman miR-34a in the miRBase 16.0 database (Griffths-Jones et al., 2006)(the mature human miR-34a sequence is SEQ ID NO:1, and its complement isSEQ ID NO:3). In either of these embodiments, the active strandcomprises the same sequence. In additional embodiments, an active strandhas a sequence that comprises or consists of SEQ ID NO:2. In someembodiments, an active strand may have modified nucleotides in which theidentity of those modified nucleotides is relative to the SEQ ID NObeing referred to.

In specific embodiments, the modified nucleotides in the active strandare the nucleotides located at positions 3 (G), 4 (C), 11 (U), 12 (U),13 (A), 14 (G), 15 (C), 16 (U), 17 (G), and/or 18 (G) relative to SEQ IDNO:1 and/or SEQ ID NO:2 (because the only difference between these twosequences is the addition of nucleotide 23, all other positions are thesame in both sequences), including any combination of modifiednucleotides thereof. This means they are the nucleotides correspondingto those nucleotides in the recited position in the recited SEQ ID NO.

It is specifically contemplated that embodiments discussed herein in thecontext of a specific nucleotide and position may also be implementedwith respect to position (from the 5′ end) only. For example, an activestrand with a modified nucleotide at position 3 (G) may also beimplemented in the context of an active strand having a modifiednucleotide at position 3 from the 5′ end of the active strand.

In specific embodiments concerning an RNA molecule, the modifiednucleotides in the active strand are the nucleotides located atpositions 3 (G), 4 (C), 11 (G), 12 (U), 13 (U), 14 (A), 15 (G), 16 (C),17 (U), and/or 18 (G) relative to SEQ ID NO:5, which contains a maturehuman miR-34c sequence.

When the particular nucleotide base is designated (as an “A,” “C,” “G,”or “U”) and is described as “relative” to a position in a sequence (suchas SEQ ID NO:2), this means that the modification of that particulardesignated nucleotide is contemplated in the strand even if its positionchanges by 1 or 2 positions (+1 or ±2 positions) (because of a deletionor insertion relative to the reference sequence). In other embodiments,a modified nucleotide is described with respect to position in thestrand and not as relative to a particular SEQ ID NO:2; in that case,position refers to the position in the strand, where the 5′ end of thestrand begins with position 1 and continues through 2, 3, 4, etc. untilthe nucleotide position at the 3′ end is reached.

An active strand comprising the sequence of SEQ ID NO:1 but notconsisting of SEQ ID NO:1 may have a nucleotide addition or insertionrelative to SEQ ID NO:1. This means if such an active strand has themodified nucleotide at position 5 relative to SEQ ID NO:1, this strandhas a modified A at position 5 in the strand or at position 6 in thestrand (depending on where the insertion or addition is). In otherwords, unless otherwise specified, modified nucleotides in the contextof a SEQ ID NO are nucleotide-specific. For example, if an active strandcomprises SEQ ID NO:1 and is 23 nucleotides in length, this means it hasan insertion relative to SEQ ID NO:1, because SEQ ID NO:1 is 22nucleotides in length. In some embodiments, if such an active strandcomprises a modified nucleotide at position 5 relative to SEQ ID NO:1,that active strand will have a modified A at position 5 in the strand ifthe insertion is after position 5, or it will have a modified A atposition 6 in the strand if the insertion is before or at position 5.

In certain embodiments, an active strand comprises the sequence of SEQID NO:5. In further embodiments, an active strand has a sequenceconsisting of the sequence in SEQ ID NO:5. In particular embodiments, anactive strand comprises the sequence of SEQ ID NO:5, but whose sequencedoes not consist of the sequence in SEQ ID NO:5.

In some embodiments, the active strand comprises at least two modifiednucleotides, though the modified nucleotides are not in the first twopositions at the 5′ end of the active strand. In certain embodiments,the active strand does not comprise a modified nucleotide in the lasttwo positions of the active strand, that is, the last two positions atthe 3′ end.

In certain embodiments, the active strand comprises a modifiednucleotide at positions 15 (C) and 16 (U) of SEQ ID NO:1 or SEQ ID NO:2.In specific embodiments, the active strand comprises a modifiednucleotide at positions 3 (G) and 4 (C) of SEQ ID NO:1 or SEQ ID NO:2,instead of, or in addition to, the modifications at positions 15 (C) and16 (U). In further embodiments, the active strand comprises a modifiednucleotide at positions 11 (U) and 12 (U) of SEQ ID NO:1 or SEQ ID NO:2,which may be instead of, or in addition to, the modifications atpositions 3 (G), 4 (C), 15 (C) and/or 16 (U). In particular embodiments,the active strand comprises a modified nucleotide at positions 11 (U),12 (U), 13(A), and 14 (G) of SEQ ID NO:1 and/or SEQ ID NO:2, which maybe instead of, or in addition to, the modifications at positions 3 (G),4 (C), 11(U), 12 (U), 15 (C) and/or 16 (U) relative to SEQ ID NO:1and/or SEQ ID NO:2. In additional embodiments, the active strandcomprises a modified nucleotide at positions 17 (G) and 18 (G) of SEQ IDNO:1 and/or SEQ ID NO:2, which may be instead of, or in addition to, themodifications at positions 3, 4, 11, 12, 13, 14, 15 and/or 16 discussedabove.

In some embodiments, the active strand has modified nucleotides atpositions 11 (U), 12 (U), 15 (C), and 16 (U) relative to SEQ ID NO:1 orSEQ ID NO:2. In other embodiments, this active strand further comprisesa modified nucleotide at positions 13 (A), 14 (G), 17 (G), and 18 (G)relative to SEQ ID NO:1 or SEQ ID NO:2.

In particular embodiments, the active strand includes a modifiednucleotide at positions 3 (G), 4 (C), 15 (C), and 16 (U) relative to SEQID NO:1 or SEQ ID NO:2.

In certain embodiments, the active strand with the sequence of SEQ IDNO:1 or SEQ ID NO:2 does not have any modified nucleotides, though insuch embodiments the passenger strand has at least a terminalmodification and at least one other modification.

In certain embodiments, the active strand is one of the followingdescribed below:

-   -   i) an active strand comprising at least two modified        nucleotides, wherein the modified nucleotides are not in the        first two positions at the 5′ end;    -   ii) an active strand comprising at least two modified        nucleotides in the first or last two positions from the ends;    -   iii) an active strand comprising modified nucleotides at        positions 3 (G), 4 (C), 11 (G), 12 (U), 13 (A), 14 (G), 15 (C),        16 (C), 17 (U), and/or 18 (G) relative to SEQ ID NO:5;    -   iv) an active strand comprising modified nucleotide at positions        11 (G) and 12 (U) relative to SEQ ID NO:5;    -   v) an active strand comprising modified nucleotides at positions        17 (U) and 18 (G) relative to SEQ ID NO:5;    -   vi) an active strand comprising modified nucleotides at        positions 11 (G), 12 (U), 17 (U), and 18 (G) relative to SEQ ID        NO:5;    -   vii) an active strand comprising modified nucleotides at        positions 3 (G) and 4 (C) and at least one modified nucleotide        at position 11 (G), 12 (U), 13 (A), 14 (G), 15 (C), 16 (C), 17        (U), and/or 18 (G) relative to SEQ ID NO:5; or,    -   viii) an active strand comprising modified nucleotides at        positions 3 (G), 4 (C), 11 (G), 12 (U), 13 (A), 14 (G), 15 (C),        16 (C), 17 (U), and 18 (G) relative to SEQ ID NO:5.

In some embodiments, RNA molecules that are double-stranded contain bothan active strand comprising all of part of the sequence of a maturemiRNA and a passenger strand fully or partially complementary to theactive strand. In some embodiments, the passenger strand is, is atleast, or is at most 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, or 100% complementary, or any rangederivable therein, to the active strand. In certain embodiments, theactive and passenger strands are fully complementary to each other.

With passenger strands containing one or more modified nucleotides, itis contemplated that there are, there are no fewer than, or there are nomore than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 modifiednucleotides, or any range derivable therein. It is specificallycontemplated that in some embodiments, fewer than every nucleotide inthe passenger strand is modified, and that fewer than half of thenucleotides in the passenger strand are modified in certain embodiments.Moreover, in some embodiments, it is specifically contemplated that apassenger strand having multiple modified nucleotides does not haveevery or every other nucleotide in the passenger strand modified.

In such embodiments, the passenger stand comprises a nucleotidemodification at the 5′ end, which may be referred to as a 5′ terminalmodification. Such a terminal modification may be with respect to thenucleotide (or nucleoside if it lacks a phosphate group) at the 5′ end.This terminal modification is specifically contemplated in someembodiments to be a modification that is not a modification of a sugarmolecule. It is specifically contemplated that this modification may beone of the following: NH₂, biotin, an amine group, a lower alkylaminegroup, NHCOCH₃, an acetyl group, 2′O-Me, DMTO, fluorescein, a thiol,acridine, Spacer 18 (PEG) amidite (DMT-Hexa(ethylene glycol)), or anyother group with this type of functionality. In specific embodiments,the 5′ terminal modification on the passenger strand is a C6 aminelinker. In further embodiments, the nucleotide at the 5′ end of thepassenger strand may have both a non-sugar modification and a sugarmodification.

In some embodiments, a passenger strand contains at least one modifiednucleotide in the first two or last two nucleotides of the passengerstrand. In certain embodiments, the passenger strand comprises amodified nucleotide in both the first two and the last two nucleotides.In other embodiments, the passenger strand has, has at least, or has atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more modifiednucleotides, or any range derivable therein. It is specificallycontemplated that in some embodiments, fewer than every nucleotide inthe passenger strand is modified, and that fewer than half of thenucleotides in the passenger strand are modified in certain embodiments.Moreover, in some embodiments, it is specifically contemplated that apassenger strand having multiple modified nucleotides does not haveevery other nucleotide in the passenger strand is modified.

In certain embodiments, the passenger strand comprises a modifiednucleotide located at positions 1 (A), 2 (A), 3 (C), 4 (A), 5 (A), 6(C), 9 (G), 10 (C), 11 (U), 12 (A), 13 (A), 14 (G), 17 (A), 18 (C), 19(U), 20 (G), 21 (C), 22 (C), and/or 23 (A) relative to SEQ ID NO:4(5′-AACAACCAGCUAAGACACUGCCA-3′) (23-mer) and any combination thereof.SEQ ID NO:4 includes the same sequence as SEQ ID NO:3(5′-ACAACCAGCUAAGACACUGCCA-3′) (22-mer) except that SEQ ID NO:4 has anadditional A at the 5′ end. In some embodiments, a passenger strandconsists of or comprises SEQ ID NO:3, but does not consist of orcomprise SEQ ID NO:4. The modified nucleotides relative to SEQ ID NO:4correspond in SEQ ID NO:3 (5′-GGCAUUCACCGCGUGCCUUA-3′) to those atpositions 1 (A), 2 (C), 3 (A), 4 (A), 5 (C), 8 (G), 9 (C), 10 (U), 11(A), 12 (A), 13 (G), 16 (A), 17 (C), 18 (U), 19 (G), 20 (C), 21 (C),and/or 22 (A).

In some embodiments, a passenger strand comprises a modified nucleotideat positions i) 1 (A) and 2 (A) and/or ii) 22 (C) and 23 (A) relative toSEQ ID NO:4. In certain embodiments, a passenger strand comprises amodified nucleotide at positions 1 (A), 2 (A), 22 (C), and 23 (A)relative to SEQ ID NO:4. In further embodiments, the passenger strandcomprises a modified nucleotide as positions i) 3 (C) and 4 (A) and/orii) 19 (U) and 20 (G) relative to SEQ ID NO:4, which may be in additionto or instead of modifications at positions iii) 1 (A), 2 (A) and/or iv)22 (C) and 23 (A) relative to SEQ ID NO:4. In certain embodiments, thepassenger strand has a modified nucleotide at positions i) 3 (C) and 4(A) or ii) 19 (U) and 20 (G) in SEQ ID NO:4, but not at both positionsi) and ii). In other embodiments, the passenger strand has modifiednucleotides at 3 (C), 4 (A), 19 (U), and 20 (G) relative to SEQ ID NO:4.In certain embodiments, the passenger strand comprises a modifiednucleotide at positions 5 (A) and 6 (C) relative to SEQ ID NO:4, whichmay be instead of or in addition to the other passenger strandmodifications discussed herein.

In other embodiments, the passenger strand comprises a modifiednucleotide at positions 9 (G) and 10 (C) relative to SEQ ID NO:4, whichmay be instead of or in addition to the other passenger strandmodifications discussed herein. In further embodiments, the passengerstrand comprises a modified nucleotide at positions 11 (U) and 12 (A)relative to SEQ ID NO:4, which may be instead of or in addition to theother passenger strand modifications discussed herein. In someembodiments, the passenger strand comprises a modified nucleotide atpositions 13 and 14 relative to SEQ ID NO:4, which may be instead of orin addition to the other passenger strand modifications discussedherein. In further embodiments, the passenger strand comprises amodified nucleotide at positions 17 (A) and 18 (C) relative to SEQ IDNO:4, which may be instead of or in addition to the other passengerstrand modifications discussed herein.

In specific embodiments, the passenger strand does not have a modifiednucleotide located at positions 7 (C), 8 (A), 9 (G), 15 (A), and 16 (C)relative to SEQ ID NO:4. In other embodiments, the passenger strandcomprises a modified nucleotide at positions 1 (A), 2 (A), 20 (G), 21(C), 22 (C), and 23 (A) relative to SEQ ID NO:4. In other embodiments,the passenger strand comprises a modified nucleotide at positions 1 (A),2 (A), 3 (C), 4 (A), 22 (C), and 23 (A) relative to SEQ ID NO:4. In someembodiments, the passenger strand comprises a modified nucleotide atpositions 1 (A), 2 (A), 19 (U), 20 (G), 22 (C), and 23 (A) relative toSEQ ID NO:4.

In certain embodiments, the passenger strand comprises a modifiednucleotide located at positions 1(G), 2 (C), 3 (A), 4 (A), 7 (A), 8 (G),9 (C), 10 (U), aa (A), 12 (A), 13 (C), 14 (U), 19 (U), 21 (C), 22 (C),and/or 23 (U) relative to SEQ ID NO:6 (5′-GCAAUCAGCUAACUACACUGCCU-3′)(23-mer) and any combination thereof.

In certain embodiments, the passenger strand comprises a modifiednucleotide located at positions 1 (G), 2 (C), 3 (A), 4 (A), 7 (A), 8(G), 9 (C), 10 (U), 11 (A), 12 (A), 13 (C), 14 (U), 19 (U), 21 (C), 22(C), and/or 23 (U) relative to SEQ ID NO:6(5′-GCAAUCAGCUAACUACACUGCCU-3′) (23-mer) and any combination thereof.

In certain embodiments, the passenger strand does not have a modifiednucleotide located at positions 5 (U), 6 (C), 15 (A), and/or 16 (C)relative to SEQ ID NO:6.

Combinations of a particular active strand and a particular passengerstrand are contemplated. It is contemplated that any active stranddescribed herein may be combined with a partially or fully complementarypassenger strand to form a double-stranded RNA molecule. In certainembodiments, there is an RNA molecule with an active strand comprisingSEQ ID NO:5 (or a sequence that has at least 90% identity with SEQ IDNO:5) and one or more modified nucleotides, and one of the followingpassenger strands:

i) a passenger strand that does not comprise any modified nucleotidesexcept a terminal modification at the 5′ end;

ii) a passenger strand comprising modified nucleotides at positions 1(G), 2 (C), 3 (A), 21 (C), 22 (C), and/or 23 (U) relative to SEQ IDNO:6;

iii) a passenger strand comprising modified nucleotides located at asubset of positions, wherein the subset is a) 1 (G), 2 (C), 3 (A) and/orb) 22 (C), and 23 (U) relative to SEQ ID NO:6;

iv) a passenger strand comprising modified nucleotides located atpositions 1 (G), 2 (C), and 3 (A) relative to SEQ ID NO:6;

v) a passenger strand comprising modified nucleotides located atpositions 22 (C) and 23 (U) relative to SEQ ID NO:6;

vi) a passenger strand comprising modified nucleotides located atpositions 1 (G), 2 (C), 3 (A), 22 (C) and 23 (U) relative to SEQ IDNO:6;

vii) a passenger strand comprising modified nucleotides in the followinggroups of positions a) and/or b), wherein a) is position 1 (G), 2 (C),and 3 (A); and b) is position 22 (C) and 23 (U) relative to SEQ ID NO:6,though in some embodiments, modified nucleotides are not located in bothgroups a) and b);

viii) a passenger strand comprising modified nucleotides located atpositions 4 (A), 7 (A), 8 (G), 9 (C), 10 (U), 11 (A), 12 (A), 13 (C), 14(U), 19 (U), and/or 21 (C) relative to SEQ ID NO:6;

ix) a passenger strand comprising modified nucleotides located atpositions 1 (G), 2 (C), 3 (A), 4 (A), 7 (A), 8 (G), 9 (C), 10 (U), 11(A), 12 (A), 13 (C), 14 (U), 19 (U), and/or 21 (C) relative to SEQ IDNO:6;

x) a passenger strand comprising modified nucleotides located atpositions 4 (A), 7 (A), 8 (G), 9 (C), 10 (U), 11 (A), 12 (A), 13 (C), 14(U), 19 (U), 21 (C) 22 (C) and/or 23 (U) relative to SEQ ID NO:6;

xi) a passenger strand comprising modified nucleotides located atpositions 1 (G), 2 (C), 3 (A), 4 (A), 7 (A), 8 (G), 9 (C), 10 (U), 11(A), 12 (A), 13 (C), 14 (U), 19 (U), 21 (C) 22 (C) and/or 23 (U)relative to SEQ ID NO:6

xii) a passenger strand comprising modified nucleotides at positions 1(G), 2 (C), 3 (A), 17 (A), 18 (C), 21 (C), 22 (C), and 23 (U) relativeto SEQ ID NO:6;

xiii) a passenger strand comprising modified nucleotides at positions 1(G), 2 (C), 3 (A), 17 (A), 18 (C), 21 (C), 22 (C), and 23 (U) relativeto SEQ ID NO:6;

xiv) a passenger strand comprising modified nucleotides at positions 1(G), 2 (C), 3 (A), 11 (A), 12 (A), 13 (C), 14 (U), 21 (C), 22 (C), and23 (U) relative to SEQ ID NO:6; or,

xv) a passenger strand comprising modified nucleotides at positions 1(G), 2 (C), 3 (A), 11 (A), 12 (A), 13 (C), 14 (U), 17 (A), 18 (C), 21(C), 22 (C), and 23 (U) relative to SEQ ID NO:6.

Combinations of a particular active strand and a particular passengerstrand are contemplated. It is contemplated that any active stranddescribed herein may be combined with any passenger strand describedherein to form a double-stranded RNA molecule.

In some embodiments, a particular combination of active and passengerstrands is contemplated for an RNA molecule that has miR-34a activity.

In certain embodiments, there is an RNA molecule with an active strandcomprising modified nucleotides at positions 11 (U), 12 (U) 15 (C), and16 (U) relative to SEQ ID NO:1 or SEQ ID NO:2 and one of the followingpassenger strands: i) a passenger strand that does not comprise anymodified nucleotides except a terminal modification at the 5′ end;

ii) a passenger strand comprising SEQ ID NO:4 and modified nucleotidesat positions 1 (A), 2 (A), 20 (G), 21 (C), 22 (C), and 23 (A) relativeto SEQ ID NO:4;

iii) a passenger strand comprising SEQ ID NO:4 and modified nucleotidesat positions 1 (A), 2 (A), 3 (C), 4 (A), 22 (C), and 23 (A) relative toSEQ ID NO:4; or,

iv) a passenger strand comprising SEQ ID NO:4 and modified nucleotidesat positions 1 (A), 2 (A), 19 (U), 20 (G), 22 (C), and 23 (A) relativeto SEQ ID NO:4.

In additional embodiments, there is an RNA molecule with an activestrand comprising modified nucleotides at positions 11 (U), 12 (U), 13(A), 14 (G), 15 (C), 16 (U), 17 (G), and 18 (G) relative to SEQ ID NO:1or SEQ ID NO:2. and one of the following passenger strands:

i) a passenger strand comprising SEQ ID NO:4 and modified nucleotides atpositions 1 (A), 2 (A), 20 (G), 21 (C), 22 (C), and 23 (A) relative toSEQ ID NO:4;

ii) a passenger strand comprising SEQ ID NO:4 and modified nucleotidesat positions 1 (A), 2 (A), 3 (C), 4 (A), 22 (C), and 23 (A) relative toSEQ ID NO:4;

iii) a passenger strand comprising SEQ ID NO:4 and modified nucleotidesat positions 1 (A), 2 (A), 19 (U), 20 (G), 22 (C), and 23 (A) relativeto SEQ ID NO:4; or

iv) a passenger strand comprising SEQ ID NO:3 or SEQ ID NO:4, wherein itdoes not comprise any modified nucleotides except a 5′ terminalmodification.

In certain embodiments, there is an RNA molecule with an active strandcomprising modified nucleotides at positions 3, 4, 15, and 16 relativeto SEQ ID NO:1 or SEQ ID NO:2 and one of the following passengerstrands:

i) a passenger strand comprising SEQ ID NO:4 and modified nucleotides atpositions 1, 2, 21, 22, and 23 relative to SEQ ID NO:4;

ii) a passenger strand comprising SEQ ID NO:4 and modified nucleotidesat positions 1, 2, 3, 4, 22, and 23 relative to SEQ ID NO:4; or,

iii) a passenger strand comprising SEQ ID NO:4 and modified nucleotidesat positions 1, 2, 19, 20, 22, and 23 relative to SEQ ID NO:4.

In other embodiments, a particular combination of active and passengerstrands is contemplated for an RNA molecule that has miR-34c activity.In certain embodiments, there is a double stranded RNA moleculecomprising a) a passenger strand comprising SEQ ID NO:6 and modifiednucleotides at positions 1 (G), 2 (C), 3 (A), 17 (A), 18 (C), 21 (C), 22(C), and 23 (U) in SEQ ID NO:6 and b) an active strand comprising SEQ IDNO:5 and modified nucleotides at positions 11 (G), 12 (U), 17 (U), and18 (G) relative to SEQ ID NO:5.

In particular embodiments, there is a double-stranded RNA moleculecomprising a) a passenger strand comprising SEQ I NO:6 and modifiednucleotides at positions 1 (G), 2 (C), 3 (A), 11 (A), 12 (A), 13 (C), 14(U), 21 (C), 22 (C), and 23 (U) in SEQ ID NO:6 and b) an active strandcomprising SEQ ID NO:5 and modified nucleotides at positions 11 (G), 12(U), 17 (U), and 18 (G) relative to SEQ ID NO:5.

In other embodiments, there is a double-stranded RNA molecule comprisinga) a passenger strand comprising SEQ ID NO:6 and modified nucleotides atpositions 1 (G), 2 (C), 3 (A), 11 (A), 12 (A), 13 (C), 14 (U), 17 (A),18 (C), 21 (C), 22 (C), and 23 (U) in SEQ ID NO:6; and b) an activestrand comprising SEQ ID NO:5 and modified nucleotides at positions 11,12, 17, and 18 in SEQ ID NO:5.

In further embodiments, there is a double-stranded RNA molecule of 22 or23 basepairs in length, wherein the RNA molecule is blunt-ended at bothends, comprising an active strand having the sequence of SEQ ID NO:5 anda separate and fully complementary passenger strand with a modifiednucleotide at the 5′ end, wherein the active strand comprises at leastone modified internal nucleotide and wherein the double-stranded RNAmolecule is more stable compared to a double-stranded, blunt-ended RNAmolecule lacking any modification of an internal nucleotide.

In certain embodiments, a miR-34 mimic is provided. In some embodiments,the mimic is a double-stranded, blunt-ended RNA molecule 22 or 23basepairs in length comprising: a) an active strand comprising i) SEQ IDNO:7 (N₁GGCAGUGUN₂N₃UUAGCUGN₄UUGN₅N₆, where N₁ is A, C, G, or U; N₂ isA, C, G, or U; N₃ is A, C, G, U, or none; N₄ is A, C, G, or U; N₅ is A,C, G, or U; and N₆ is A, C, G, U, or none) from 5′ to 3′ and ii) atleast one modified internal nucleotide; and, b) a fully complementarypassenger strand comprising a terminal modification of the nucleotide atthe 5′ end. In particular embodiments, the molecule is 23 basepairs inlength.

In further embodiments, an RNA molecule comprising SEQ ID NO:7 has anactive strand with a modified nucleotide at position 3, 4, 11, 12, 13,14, 15, 16, 17, and/or 18. This designation is position-specific (notnucleotide-specific) with respect to the active strand. As with SEQ IDNO:2 (mir-34a) and SEQ ID NO:5 (miR-34c), in some embodiments, an RNAmolecule has one of the following active strands:

i) an active strand comprising at least two modified nucleotides,wherein the modified nucleotides are not in the first two positions atthe 5′ end;

ii) an active strand comprising at least two modified nucleotides in thefirst or last two positions from the ends;

iii) an active strand comprising modified nucleotides at positions 3, 4,11, 12, 13, 14, 15, 16, 17, and/or 18;

iv) an active strand comprising modified nucleotide at positions 11 and12;

v) an active strand comprising modified nucleotides at positions 17 and18;

vi) an active strand comprising modified nucleotides at positions 11,12, 17, and 18;

vii) an active strand comprising modified nucleotides at positions 3 (G)and 4 (C) and at least one modified nucleotide at position 11, 12, 13,14, 15, 16, 17, and/or 18;

viii) an active strand comprising modified nucleotides at positions 3,4, 11, 12, 13, 14, 15, 16, 17, and 18; or,

ix) an active strand comprising no more than 11 modified internalnucleotides.

In further embodiments, the nucleotide modifications discussed in thecontext of either SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9may be implemented in the context of any other of SEQ ID NO:2, SEQ IDNO:5, SEQ ID NO:7, or SEQ ID NO:9. These embodiments that may beimplemented in the context of one of these SEQ ID NOs may describe anucleotide modification that is nucleotide-based or position-based. SEQID NO:9 includes SEQ ID NO:7, which includes SEQ ID NO:2 and SEQ IDNO:5. All of these concern active strands discussed herein.

In certain embodiments, an active strand comprises SEQ ID NO:5. Incertain embodiments comprising an RNA molecule with an active strandcomprising SEQ ID NO:7, the passenger strand comprises at least onemodified internal nucleotide, in addition to a terminalnucleotide/nucleoside modification. In some embodiments, a passengerstrand comprises a modified nucleotide at position 1, 2, 3, 4, 9, 10,11, 12, 13, 14, 19, 21, and/or 22. In certain embodiments, the passengerstrand does not have a modified nucleotide at position 15 and/or 16.

In some embodiments, the passenger strand comprises SEQ ID NO:3. Incertain embodiments, a passenger strand comprises a modified nucleotideat position 1, 2, 3, 5, 6, 9, 10, 11, 12, 13, 14, 17, 18, 19, 20, 21,and/or 22 in the passenger strand. In other embodiments, a passengerstrand comprises SEQ ID NO:4. In additional embodiments, a passengerstrand comprises a modified nucleotide at position 1, 2, 3, 4, 7, 8, 9,10, 11, 12, 13, 14, 19, 21, 22, and/or 23.

In further embodiments, the nucleotide modifications discussed in thecontext of either SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10may be implemented in the context of any other of SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, or SEQ ID NO:10. These embodiments that may beimplemented in the context of one of these SEQ ID NOs may describe anucleotide modification that is nucleotide-based or position-based. SEQID NO:10 includes SEQ ID NO:8, which includes SEQ ID NO:4 and SEQ IDNO:6. All of these concern passenger strands discussed

In certain embodiments, there is an miR-34 mimic having SEQ ID NO:7. Insuch RNA molecules, there are embodiments in which an active strand hasone or more modified nucleotides, wherein the modified nucleotide isidentified as being nucleotide-specific. In such embodiments, there isan active strand comprising modified nucleotides at positions 3 (G), 4(C), 11 (G), 12 (U), 13 (A), 14 (G), 15 (C), 16 (C), 17 (U), and/or 18(G) relative to SEQ ID NO:7; in other embodiments, the active strandcomprises modified nucleotide at positions 11 (G) and 12 (U) relative toSEQ ID NO:7; in additional embodiments an active strand comprisesmodified nucleotides at positions 17 (U) and 18 (G) relative to SEQ IDNO:7; in additional embodiments, an active strand comprises modifiednucleotides at positions 11 (G), 12 (U), 17 (U), and 18 (G) relative toSEQ ID NO:7; in further embodiments, an active strand comprises modifiednucleotides at positions 3 (G) and 4 (C) and at least one modifiednucleotide at position 11 (G), 12 (U), 13 (A), 14 (G), 15 (C), 16 (C),17 (U), and/or 18 (G) relative to SEQ ID NO:7; and in even furtherembodiments, an active strand comprises modified nucleotides atpositions 3 (G), 4 (C), 11 (G), 12 (U), 13 (A), 14 (G), 15 (C), 16 (C),17 (U), and 18 (G) relative to SEQ ID NO:7.

In certain embodiments, this combination of active and passenger strandshas a 5′ terminal modification of the passenger strand in which theterminal modification is an alkyl amine such as a C6 amine linker, andthe nucleotide modifications are on the sugar at the 2′ position. Inspecific embodiments, the sugar modification is a 2′OMe.

In further embodiments, there is a double-stranded RNA molecule of 22 or23 basepairs in length, wherein the RNA molecule is blunt-ended at bothends, comprising an active strand having the sequence of SEQ ID NO:1 anda separate and fully complementary passenger strand with a modifiednucleotide at the 5′ end, wherein the active strand comprises at leastone modified internal nucleotide and wherein the double-stranded RNAmolecule is more stable in the presence of a nuclease compared to adouble-stranded, blunt-ended RNA molecule lacking any modification of aninternal nucleotide.

In some embodiments, the RNA molecule has nucleotides that are modifiedwith a sugar modification. In specific embodiments, the sugarmodification is 2′-OMe.

Specific embodiments include pharmaceutical compositions containing oneor more different RNA molecules capable of acting as miRNA mimics; thedifference may relate to sequence and/or type or position ofmodification. In certain embodiments, the RNA molecules are comprised ina lipid formulation. In other embodiments, RNA molecules may beformulated with a liposome, polymer-based nanoparticle, cholesterolconjugate, cyclodextran complex, polyethylenimine polymer and/or aprotein complex.

Methods for providing miR-34a activity to a cell are also set forth inembodiments. In some embodiments, there are methods for providingmiR-34a activity to a cell comprising administering to the cell aneffective amount of an RNA molecule having miR-34a activity. In someembodiments, the cell is a cancer cell. Such RNA molecules are discussedthroughout this disclosure.

Other methods include a method for decreasing cell proliferationcomprising administering to the cell an effective amount of a miR-34amimic. Additional embodiments include methods for inducing apoptosis ina cell comprising administering to the cell an effective amount of themiR-34a mimic. Other embodiments concern methods for treating cancer ina patient comprising administering to the patient a pharmaceuticalcomposition comprising one or more of the RNA molecules that have miRNAfunction. Further embodiments concern methods of inhibiting progressionthrough cell cycle by administering an effective amount of a miR-34amimic discussed herein. In some embodiments, methods further compriseadministering to the patient an additional cancer therapy. In someembodiments, a patient has been tested for and/or diagnosed with cancer.Such methods may involve the double-stranded RNA molecules discussedherein. Moreover, it is contemplated that multiple different miR-34amimics may be employed in methods and compositions discussed herein.

Other embodiments concern the use of RNA molecules for treating cancercells, or their use in decreasing cell proliferation, inducing apoptosisor providing miR-34a function to a cell.

Methods for providing miR-34c activity to a cell are also set forth inembodiments. In some embodiments, there are methods for providingmiR-34c activity to a cell comprising administering to the cell aneffective amount of an RNA molecule having miR-34c activity. In someembodiments, the cell is a cancer cell. Such RNA molecules are discussedthroughout this disclosure.

Other methods include a method for decreasing cell proliferationcomprising administering to the cell an effective amount of a miR-34cmimic. Additional embodiments include methods for inducing apoptosis ina cell comprising administering to the cell an effective amount of themiR-34c mimic. Other embodiments concern methods for treating cancer ina patient comprising administering to the patient a pharmaceuticalcomposition comprising the RNA molecules that have miRNA function. Insome embodiments, methods further comprise administering to the patientan additional cancer therapy. In some embodiments, a patient has beentested for and/or diagnosed with cancer. Such methods may involve thedouble-stranded RNA molecules discussed herein. Moreover, it iscontemplated that multiple different miR-34c mimics may be employed inmethods and compositions discussed herein.

Other embodiments concern the use of RNA molecules for treating cancercells, or their use in decreasing cell proliferation, inducing apoptosisor to provide miR-34c function to a cell.

Methods discussed in the context of a miR-34a mimic may be implementedwith miR-34c mimic, and vice versa.

The compositions and methods for their use can “comprise,” “consistessentially of,” or “consist of” any of the molecules or steps disclosedthroughout the specification. With respect to the transitional phase“consisting essentially of,” and in one non-limiting aspect, a basic andnovel characteristic of the compositions and methods disclosed in thisspecification includes the miRNA mimic activity.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

It is contemplated that any embodiment discussed herein can beimplemented with respect to any method or composition of the invention,and vice versa. Furthermore, compositions and kits of the invention canbe used to achieve methods of the invention.

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” It is also contemplatedthat anything listed using the term “or” may also be specificallyexcluded.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments are directed to compositions and methods relating to miRNAs,as well as use of miRNA mimics. Methods include preparing such mimicsand using such mimics to provide miRNA activity or function to a cell.In certain embodiments, miRNA mimics are used for therapeutic,prognostic, and diagnostic applications, particularly those methods andcompositions related to therapeutic applications for conditions ordiseases in which miRNA activity or function is involved.

I. NUCLEIC ACIDS

Nucleic acids include the sequences or segments of sequence that areidentical, complementary, or partially complementary sequences to maturemicroRNA (“miRNA” or “miR”) molecules. Mature miRNA molecules aregenerally 21 to 22 nucleotides in length, though lengths of 16 and up to27 nucleotides have been reported. The miRNAs are each processed from alonger precursor RNA molecule (“precursor miRNA”). Precursor miRNAs aretranscribed from non-protein-encoding genes. The precursor miRNAs havetwo regions of complementarity that enables them to form a stem-loop- orfold-back-like structure, which is cleaved in animals by a ribonucleaseIII-like nuclease enzyme called Dicer. The processed miRNA is typicallya portion of the stem.

The processed miRNA (also referred to as “mature miRNA”) becomes part ofa large complex to down-regulate a particular target gene. Examples ofanimal miRNAs include those that imperfectly basepair with the target,which halts translation (Olsen et al., 1999; Seggerson et al., 2002).siRNA molecules also are processed by Dicer, but from a long,double-stranded RNA molecule. siRNAs are not naturally found in animalcells, but they can direct the sequence-specific cleavage of an mRNAtarget through an RNA-induced silencing complex (RISC) (Denli et al.,2003).

A. miR-34a and miR-34c

It was previously demonstrated that miR-34 is involved with theregulation of numerous cell activities that represent interventionpoints for cancer therapy and for therapy of other diseases anddisorders (U.S. patent application Ser. No. 11/141,707 filed May 31,2005 and Ser. No. 11/273,640 filed Nov. 14, 2005), which are herebyincorporated by reference.

The inventors also demonstrated that miR-34 functions as a tumorsuppressor through its ability to regulate the expression of a numbersof key oncogenes (U.S. patent application Ser. No. 12/134,932 filed Jun.6, 2008, which is hereby incorporated by reference). Among thecancer-related genes that are regulated directly or indirectly by miR-34are Angiogenin, aurora kinase B, BCL10, BRCA1, BRCA2, BUB1, cyclin A2,cyclin D1, cyclin D3, CDK-4, CDK inhibitor 2C, FAS, forkhead box M1,HDAC-1, c-Jun, MCAM, Mcl-1, c-Met, Myb L2, NF1, NF2, PI 3-kinase,polo-like kinase 1, R-RAS, SMAD3, TGF beta receptor, TPD52 tumor proteinD52, and Wnt-7b.

In summary, miR-34 governs the activity of proteins that are criticalregulators of cell proliferation and survival. These targets arefrequently deregulated in human cancer.

B. Oligomeric Compounds

Embodiments concern miRNA mimics, which contain molecules capable ofmimicking the activity of an RNA molecule. An RNA molecule contains anucleoside, which is a base-sugar combination. The base portion of thenucleoside is typically a heterocyclic base moiety. The two most commonclasses of such heterocyclic bases are purines and pyrimidines.Nucleotides are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar. It iscontemplated that an RNA strand will be composed of nucleotides(ribonucleotides) and that the 5′ end may be a nucleotide or anucleoside. In other words, there may be a phosphate group linked to thesugar portion of the nucleoside or there may be only a hydroxyl groupinstead of the phosphate group. As discussed herein, in someembodiments, there is a modification of a terminal nucleoside ornucleotide in which a chemical moiety or group is attached to the sugarthrough what is, or was formerly, a hydroxyl or phosphate group.

In forming oligonucleotides, the phosphate groups covalently linkadjacent nucleosides to one another to form a linear polymeric compound.The respective ends of this linear polymeric structure can be joined toform a circular structure by hybridization or by formation of a covalentbond. In addition, linear compounds may have internal nucleobasecomplementarity and may therefore fold in a manner as to produce a fullyor partially double-stranded structure. Within the oligonucleotidestructure, the phosphate groups are commonly referred to as forming theinternucleoside linkages of the oligonucleotide. The normalinternucleoside linkage of RNA and DNA is a 3′ to 5′ phosphodiesterlinkage.

In some embodiments, there is an RNA, RNA molecule, or RNA analog havinga length of between 17 and 130 residues. Embodiments concern syntheticmiRNA molecules that are, are at least, or are at most 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, or 40 or more residues in length, including any integer orany range derivable therein. Each strand or RNA molecule in adouble-stranded RNA molecule may be such lengths as recited above. Insome embodiments, an RNA molecule has a blunt end on one or both ends.In certain embodiments, the RNA molecule has a blunt end on the sidehaving the 5′ end of the active strand. In other embodiments, the RNAmolecule has a blunt end on the side having the 5′ end of the passengerstrand.

RNA molecules described herein may have one or two strands. In moleculeswith two strands, the two strands may be hybridized to one another, butthey are not connected to one another by an internucleoside linkage.

In certain embodiments, such RNA molecules that comprise or consist ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9 (orthat consists of a sequence that has at least 90% identity with one ofthe recited SEQ ID NOs) have a modified nucleotide or nucleoside locatedat position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, and/or 23 in the active strand (position 1 is the 5′end). In further embodiments, a modified nucleotide or nucleoside islocated at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, and/or 23 in a passenger strand (position 1is the 5′ end) that comprises or consists of SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10 (or that consists of asequence that has at least 90% identity with one of the recited SEQ IDNOs). The designation of the modified nucleotide is position-specific,as opposed to nucleotide-specific. Accordingly, an embodiment in whichnucleotide-specific modifications are discussed, for example, “an activestrand comprising modified nucleotide at positions 11 (G) and 12 (U)relative to SEQ ID NO:5,” may be implemented in other embodiments withrespect to position; consequently, in further embodiments, an RNAmolecule may comprise, for example, an strand comprising a modifiednucleotide at positions 11 and 12.

In some embodiments, the miRNA mimic or RNA molecule is not blunt-endedon both sides. It is contemplated that there may be a 1, 2, 3, 4, 5, or6 base overhang on either the 3′ or 5′ end of the passenger or activestrands of a double-stranded RNA mimic or molecule.

In some embodiments, the passenger strand and the active strand are notfully complementary. It is contemplated that there may be 1, 2, 3, 4, 5,6 or more nucleotides between the two strands that are notcomplementary. In some embodiments, these nucleotides are within thefirst 10 nucleotides of the 5′ end of the passenger strand.

It is contemplated that RNA mimics have RNA bases, which may or may notbe modified. As such, RNA mimics are RNA or RNA molecules. Moreover, itis understood that a nucleic acid, including RNA, may have more thanone-strand. As discussed herein, in some embodiments a miRNA mimic orRNA molecule is double-stranded. Unless otherwise specified, adouble-stranded RNA molecule or miRNA mimic will be understood to havetwo strands that can be separated from each other and that are notsimply connected to one another by a hairpin linker. A hairpin moleculehas one strand that is capable of intramolecular hybridization. In someembodiments, the miRNA mimic is a hairpin molecule. In others, the miRNAmimic is a double-stranded RNA molecule.

In certain embodiments, therapeutic double-stranded nucleic acids have afirst active strand with (a) a “miRNA region” whose sequence from 5′ to3′ is identical to all or a segment of a mature miRNA sequence, and asecond passenger strand having (b) a “complementary region” whosesequence from 5′ to 3′ is between 60% and 100% complementary to themiRNA sequence. In certain embodiments, these synthetic miRNA are alsoisolated, as defined below, or purified. The term “miRNA region” refersto a region on the synthetic miRNA that is at least 75, 80, 85, 90, 95,or 100% identical, including all integers there between, to the entiresequence of a mature, naturally occurring miRNA sequence. In certainembodiments, the miRNA region is or is at least 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or100% identical to the sequence of a naturally-occurring miRNA, such asthe human miRNA sequence. Alternatively, the miRNA region can comprise18, 19, 20, 21, 22, 23, 24 or more nucleotide positions in common with anaturally-occurring miRNA as compared by sequence alignment algorithmsand methods well known in the art.

The term “complementary region” refers to a region of a synthetic miRNAthat is or is at least 60% complementary to the mature, naturallyoccurring miRNA sequence that the miRNA region is identical to. Thecomplementary region is or is at least 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2,99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% complementary, or anyrange derivable therein. With single polynucleotide sequences, there maybe a hairpin loop structure as a result of chemical bonding between themiRNA region and the complementary region. In other embodiments, thecomplementary region is on a different nucleic acid strand than themiRNA region, in which case the complementary region is on the passengerstrand and the miRNA region is on the active strand.

The term “oligonucleotide” is understood in the art to refer to anoligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid(DNA), typically one that is no more than 100 bases or base pairs inlength. It is contemplated that an oligonucleotide may have a nucleosideat the 5′ end. This term includes oligonucleotides composed of naturallyoccurring nucleobases, sugars and covalent internucleoside linkages. Theterm “oligonucleotide analog” refers to oligonucleotides that have oneor more non-naturally occurring portions which function in a similarmanner to oligonucleotides. Such non-naturally occurringoligonucleotides may have desirable properties compared to the naturallyoccurring oligonucleotides such as, for example, those disclosed herein,including, but not limited to, increased physiological activity,increased stability in the presence of a nuclease(s), and/or increasedpharmacokinetic properties.

The term “oligonucleoside” refers to nucleosides that are chemicallyconnected via internucleoside linkages that do not have phosphorusatoms. Internucleoside linkages include short chain alkyl, cycloalkyl,mixed heteroatom alkyl, mixed heteroatom cycloalkyl, one or more shortchain heteroatomic and one or more short chain heterocyclic. Theseinternucleoside linkages include, but are not limited to, siloxane,sulfide, sulfoxide, sulfone, acetyl, formacetyl, thioformacetyl,methylene formacetyl, thioformacetyl, alkeneyl, sulfamate;methyleneimino, methylenehydrazino, sulfonate, sulfonamide, amide andothers having mixed N, O, S and CH₂ component parts. In addition to themodifications described above, the nucleosides of the oligomericcompounds of the invention can have a variety of other modifications.Additional nucleosides amenable to embodiments having modified basemoieties and or modified sugar moieties are disclosed in U.S. Pat. No.6,383,808 and PCT application PCT/US89/02323, both of which are herebyincorporated by reference.

Altered base moieties or altered sugar moieties also include othermodifications consistent with the purpose of an miRNA mimic. Sucholigomeric compounds are best described as being structurallydistinguishable from, yet functionally interchangeable with, naturallyoccurring or synthetic unmodified oligonucleotides. All such oligomericcompounds are comprehended by this invention so long as they functioneffectively to mimic the structure or function of a desired RNA or DNAoligonucleotide strand.

In some embodiments, RNA mimics include a base modification orsubstitution. The natural or unmodified bases in RNA are adenine (A) andguanine (G), and the pyrimidine bases cytosine (C) and uracil (U) (DNAhas thymine (T)). In contrast, modified bases, also referred to asheterocyclic base moieties, include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine and otheralkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo (including 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines), 7-methylguanine and7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine.

One or more base or sugar modifications may be used to induce a 3′-endosugar conformation. A nucleoside can incorporate synthetic modificationsof the heterocyclic base, the sugar moiety or both to induce a desired3′-endo sugar conformation. These modified nucleosides are used to mimicRNA-like nucleosides so that particular properties of an oligomericcompound can be enhanced while maintaining the desired 3′-endoconformational geometry (see Scheme 1 of U.S. Patent ApplicationPublication 2005/0261218, which is hereby incorporated by reference).

In some embodiments, an RNA mimic has a modification particularly of the5′ terminal residue of specifically the strand of an RNA mimic havingthe sequence that is complementary to the mature miRNA. This strand isreferred to as the “passenger” strand herein. Without being bound totheory, it appears that the presence of a stable moiety other than aphosphate or hydroxyl at the 5′ end of the complementary strand impairsor eliminates uptake of the passenger strand by the miRNA pathwaycomplex and subsequently favors uptake of the active strand by the miRNAprotein complex. 5′ modifications include, but are not limited to, NH₂,biotin, an amine group, a lower alkylamine group, a lower alkyl group,NHCOCH₃, an acetyl group, 2′oxygen-methyl (2′O-Me), DMTO, fluorescein, athiol, or acridine or any other group with this type of functionality.In other embodiments, there is a Spacer 18 (PEG) amidite(DMT-Hexa(ethylene glycol)). In other embodiments, there is analkylamine or alkyl group of 40 carbons or fewer. In embodimentsinvolving a “lower” alkylamine or alkyl group, “lower” will beunderstood to refer to a molecule with 20 or fewer carbons.

In specific embodiments, there is a C4-C12 amine linker on the 5′ end ofthe passenger strand. In specific embodiments, there is a C6 amine onthe terminal phosphate of the first nucleotide of the passenger strand:

In specific embodiments, there is a C12 amine linker on the 5′ end ofthe passenger strand. In other embodiments, there is a C8 amine linkeron the terminal phosphate of the first nucleotide of the passengerstrand.

In different miRNA mimics discussed herein, these RNA molecules can havenucleotides with sugar portions that correspond to naturally occurringsugars or modified sugars. Representative modified sugars includecarbocyclic or acyclic sugars, sugars having substituent groups at oneor more of their 2′, 3′ or 4′ positions and sugars having substituentsin place of one or more hydrogen atoms of the sugar. In certainembodiments, the sugar is modified by having a substituent group at the2′ position. In additional embodiments, the sugar is modified by havinga substituent group at the 3′ position. In other embodiments, the sugaris modified by having a substituent group at the 4′ position. It is alsocontemplated that a sugar may have a modification at more than one ofthose positions, or that an RNA molecule may have one or morenucleotides with a sugar modification at one position and also one ormore nucleotides with a sugar modification at a different position.

Sugar modifications contemplated in miRNA mimics include, but are notlimited to, a sugar substituent group selected from: OH; F; O-, S-, orN-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl,wherein the alkyl, alkenyl and alkynyl may be substituted orunsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. In someembodiments, these groups may be chosen from: O(CH₂)_(x)OCH₃,O((CH₂)_(x)O)_(y)CH₃, O(CH₂)_(x)NH₂, O(CH₂)_(x)CH₃, O(CH₂)_(x)ONH₂, andO(CH₂)_(x)ON((CH₂)_(x)CH₃)₂, where x and y are from 1 to 10.

In some embodiments, miRNA mimics have a sugar substituent groupselected from the following: C1 to C₁₀ lower alkyl, substituted loweralkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH₃, Cl, Br, CN, OCN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of amimic, or a group for improving the pharmacodynamic properties of amimic, and other substituents having similar properties. In oneembodiment, the modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃,which is also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al.,1995), that is, an alkoxyalkoxy group. Another modification includes2′-dimethylaminooxyethoxy, that is, a O(CH₂)₂ON(CH₃)₂ group, also knownas 2′-DMAOE and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), that is,2′-O—CH₂—O—CH₂—N(CH₃)₂.

Additional sugar substituent groups include allyl (—CH₂—CH═CH₂),—O-allyl CH₂—CH═CH₂), methoxy (—O—CH₃), aminopropoxy (—OCH₂CH₂CH₂NH₂),and fluoro (F). Sugar substituent groups on the 2′ position (2′-) may bein the arabino (up) position or ribo (down) position. One 2′-arabinomodification is 2′-F. Other similar modifications may also be made atother positions on the oligomeric compound, particularly the 3′ positionof the sugar on the 3′ terminal nucleoside or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligomeric compounds may also have sugar mimetics, for example,cyclobutyl moieties, in place of the pentofuranosyl sugar. Examples ofU.S. patents that disclose the preparation of modified sugar structuresinclude, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800;5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300;5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and5,700,920, which are herein incorporated by reference in its entirety.

Representative sugar substituent groups include groups described in U.S.Patent Application Publication 2005/0261218, which is herebyincorporated by reference. In particular embodiments, the sugarmodification is a 2′O-Me modification, a 2′F modification, a 2′Hmodification, a 2′amino modification, a 4′thioribose modification or aphosphorothioate modification on the carboxy group linked to the carbonat position 6′, or combinations thereof.

Additional modifications are disclosed in U.S. Patent ApplicationPublication 2010/0267814, which is hereby incorporated by reference.While this references discloses general modifications that might bemade, it does not disclose what is set forth herein that modificationsmight be made in the context of a particular sequence at specificnucleotides and/or in specific and select positions.

In some embodiments, a therapeutic nucleic acid contains one or moredesign elements. These design elements include, but are not limited to:(i) a replacement group for the phosphate or hydroxyl of the nucleotideor nucleoside, respectively, at the 5′ terminus of the complementaryregion; (ii) one or more sugar modifications in the first or last 1 to 6residues of the complementary region; or, (iii) non-complementaritybetween one or more nucleotides in the last 1 to 5 residues at the 3′end of the complementary region and the corresponding nucleotides of themiRNA region.

In certain embodiments, a synthetic miRNA has a nucleotide at its 5′ endof the complementary region in which the phosphate and/or hydroxyl grouphas been replaced with another chemical group (referred to as the“replacement design”). In some cases, the phosphate group is replaced oradded onto with an additional moiety, while in others, the hydroxylgroup has been replaced or added onto with an additional moiety, such asdescribed above with the C6 amine linker. In particular embodiments, themoiety is biotin, an amine group, a lower alkylamine group, an acetylgroup, 2′O-Me (2′ oxygen-methyl), DMTO (4,4′-dimethoxytrityl withoxygen), fluorescein, a thiol, or acridine, though other moieties arewell known to those of skill in the art and can be used as well.

In other embodiments of the invention, there is a synthetic miRNA inwhich one or more nucleotides in the last 1 to 5 residues at the 3′ endof the complementary region are not complementary to the correspondingnucleotides of the miRNA region (“non-complementarity”) (referred to asthe “non-complementarity design”). The non-complementarity may be in thelast 1, 2, 3, 4, and/or 5 residues of the complementary miRNA. Incertain embodiments, there is non-complementarity with at least 2nucleotides in the complementary region.

It is contemplated that synthetic miRNA of the invention have one ormore of the replacement, sugar modification, or non-complementaritydesigns. In certain cases, synthetic RNA molecules have two of them,while in others these molecules have all three designs in place.

The miRNA region and the complementary region may be on the same orseparate polynucleotides. In cases in which they are contained on or inthe same polynucleotide, the miRNA molecule will be considered a singlepolynucleotide. In embodiments in which the different regions are onseparate polynucleotides, the synthetic miRNA will be considered to becomprised of two polynucleotides.

When the RNA molecule is a single polynucleotide, there is a linkerregion between the miRNA region and the complementary region. In someembodiments, the single polynucleotide is capable of forming a hairpinloop structure as a result of bonding between the miRNA region and thecomplementary region. The linker constitutes the hairpin loop. It iscontemplated that in some embodiments, the linker region is, is atleast, or is at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, or 40 residues in length, or any range derivabletherein. In certain embodiments, the linker is between 3 and 30 residues(inclusive) in length.

In addition to having a miRNA region and a complementary region, theremay be flanking sequences as well at either the 5′ or 3′ end of theregion. In some embodiments, there is or is at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10 nucleotides or more, or any range derivable therein,flanking one or both sides of these regions.

RNA molecules with miRNA function may be, be at least, or be at most 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240,250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380,390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520,530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660,670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800,810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940,950, 960, 970, 980, 990, or 1000 nucleotides, or any range derivabletherein, in length. Such lengths cover the lengths of processed miRNA,miRNA probes, precursor miRNA, miRNA containing vectors, control nucleicacids, and other probes and primers. In many embodiments, miRNA are19-24 nucleotides in length, while miRNA probes are 5, 10, 15, 20, 25,30, to 35 nucleotides in length, including all values and ranges therebetween, depending on the length of the processed miRNA and any flankingregions added. miRNA precursors are generally between 62 and 110nucleotides in humans.

Nucleic acids of the invention may have regions of identity orcomplementarity to another nucleic acid. It is contemplated that theregion of complementarity or identity can be at least 5 contiguousresidues, though it is specifically contemplated that the region is, isat least, or is at most 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or 110 contiguous nucleotides.It is further understood that the length of complementarity within aprecursor miRNA or between a miRNA probe and a miRNA or a miRNA gene aresuch lengths. Moreover, the complementarity may be expressed as apercentage, meaning that the complementarity between a probe and itstarget is 90% identical or greater over the length of the probe. In someembodiments, complementarity is or is at least 90%, 95% or 100%identical. In particular, such lengths may be applied to any nucleicacid comprising a nucleic acid sequence identified in any of SEQ ID NOsdisclosed herein.

The term “recombinant” may be used and this generally refers to amolecule that has been manipulated in vitro or that is a replicated orexpressed product of such a molecule.

The term “miRNA” generally refers to an RNA molecule having a sequenceand function of an miRNA molecule. In specific embodiments, moleculesimplemented in the invention will also encompass a region or anadditional strand that is partially (between 10 and 50% complementaryacross length of strand), substantially (greater than 50% but less than100% complementary across length of strand) or fully complementary toanother region of the same single-stranded molecule or to anothernucleic acid. Thus, nucleic acids may encompass a molecule thatcomprises one or more complementary or self-complementary strand(s) or“complement(s)” of a particular sequence comprising a molecule. Forexample, precursor miRNA may have a self-complementary region, which isup to 100% complementary. miRNA probes or nucleic acids of the inventioncan include, can be or can be at least 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98, 99 or 100% complementary to their target.

Nucleic acids of the invention may be made by any technique known to oneof ordinary skill in the art, such as for example, chemical synthesis,enzymatic production or biological production. It is specificallycontemplated that miRNA probes of the invention are chemicallysynthesized.

In some embodiments of the invention, miRNAs are recovered or isolatedfrom a biological sample. The miRNA may be recombinant or it may benatural or endogenous to the cell (produced from the cell's genome). Itis contemplated that a biological sample may be treated in a way so asto enhance the recovery of small RNA molecules such as miRNA. U.S.patent application Ser. No. 10/667,126 describes such methods and it isspecifically incorporated by reference herein. Generally, methodsinvolve lysing cells with a solution having guanidinium and a detergent.

In certain aspects, synthetic miRNA are RNA or RNA analogs. miRNA mimicsmay be DNA and/or RNA, or analogs thereof. miRNA mimics with chemicalmodifications may be collectively referred to as “synthetic nucleicacids.”

In some embodiments, a therapeutic nucleic acid can have a miRNA or asynthetic miRNA sequence of between 10-200 to between 17-130 residues,including all values and ranges there between. The present inventionconcerns miRNA or synthetic miRNA molecules that are, are at least, orare at most 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 140, 150, 160, 170, 180, 190, 200 or more residues in length,including any integer or any range there between.

In certain aspects, synthetic nucleic acids have (a) a “miRNA region”whose sequence or binding region from 5′ to 3′ is identical orcomplementary to all or a segment of a mature miRNA sequence, and (b) a“complementary region” whose sequence from 5′ to 3′ is between 60% and100% complementary to the miRNA sequence in (a). In certain embodiments,these synthetic nucleic acids are also isolated, as defined below. Theterm “miRNA region” refers to a region on the synthetic nucleic acidthat is at least 75, 80, 85, 90, 95, or 100% identical, including allintegers there between, to the entire sequence of a mature, naturallyoccurring miRNA sequence or a complement thereof. In certainembodiments, the miRNA region is or is at least 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or100% identical to the sequence of a naturally-occurring miRNA, orsegment thereof, or complement thereof.

Discussed herein are embodiments involving miR-34 mimics, includingspecific miR-34a and miR-34c mimics. Different active and passengerstrands for these mimics are described throughout the disclosure. It iscontemplated that embodiments discussed in the context of a particularSEQ ID NO may be implemented in addition to or instead of otherembodiments discussing the same SEQ ID NO. For example, an active strandthat has at least 90% identity to SEQ ID NO:5 and also has asubstitution of one of the nucleotides/nucleoside may be combined withan embodiment of an active strand involving SEQ ID NO:5 that also has aninsertion in the sequence; accordingly, an active strand that has atleast 90% identity to SEQ ID NO:5 would have both a substitution and aninsertion relative to SEQ ID NO:5.

In embodiments concerning an miRNA-34a mimic, it is contemplated that anRNA molecule may contain an active strand that is or is at least 90, 91,92, 93, 94, 95, 96, 97, 98, 99 or 100% identical, or any range derivabletherein, to SEQ ID NO:1. In other embodiments, the active strand is oris at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical, orany range derivable therein, to SEQ ID NO:2.

In some embodiments, an active strand is 95% identical to SEQ ID NO:2(5′-UGGCAGUGUCUUAGCUGGUUGUU-3′) (23-mer). In certain embodiments, theactive strand has the following sequence from 5′ to 3′ in which onenucleotide from SEQ ID NO:2 is deleted:

UGGCAGUGUCUUAGCUGGUUGU (SEQ ID NO: 1)(U formerly at position 23 deleted) UGGCAGUGUCUUAGCUGGUUGU(SEQ ID NO: 1) (U formerly at position 22 deleted)UGGCAGUGUCUUAGCUGGUUUU (SEQ ID NO: 11)(G formerly at position 21 deleted) UGGCAGUGUCUUAGCUGGUGUU(SEQ ID NO: 12) (U formerly at position 20 deleted)UGGCAGUGUCUUAGCUGGUGUU (SEQ ID NO: 12)(U formerly at position 19 deleted) UGGCAGUGUCUUAGCUGUUGUU(SEQ ID NO: 13) (G formerly at position 18 deleted)UGGCAGUGUCUUAGCUGUUGUU (SEQ ID NO: 13)(G formerly at position 17 deleted) UGGCAGUGUCUUAGCGGUUGUU(SEQ ID NO: 14) (U formerly at position 16 deleted)UGGCAGUGUCUUAGUGGUUGUU (SEQ ID NO: 15)(C formerly at position 15 deleted) UGGCAGUGUCUUACUGGUUGUU(SEQ ID NO: 16) (G formerly at position 14 deleted)UGGCAGUGUCUUGCUGGUUGUU (SEQ ID NO: 17)(A formerly at position 13 deleted) UGGCAGUGUCUAGCUGGUUGUU(SEQ ID NO: 18) (U formerly at position 12 deleted)UGGCAGUGUCUAGCUGGUUGUU (SEQ ID NO: 18)(U formerly at position 11 deleted) UGGCAGUGUUUAGCUGGUUGUU(SEQ ID NO: 19) (C formerly at position 10 deleted)UGGCAGUGCUUAGCUGGUUGUU (SEQ ID NO: 20)(U formerly at position 9 deleted) UGGCAGUUCUUAGCUGGUUGUU(SEQ ID NO: 21) (G formerly at position 8 deleted)UGGCAGGUCUUAGCUGGUUGUU (SEQ ID NO: 22)(U formerly at position 7 deleted) UGGCAUGUCUUAGCUGGUUGUU(SEQ ID NO: 23) (G formerly at position 6 deleted)UGGCGUGUCUUAGCUGGUUGUU (SEQ ID NO: 24)(A formerly at position 5 deleted) UGGAGUGUCUUAGCUGGUUGUU(SEQ ID NO: 25) (C formerly at position 4 deleted)UGCAGUGUCUUAGCUGGUUGUU (SEQ ID NO: 26)(G formerly at position 3 deleted) UGCAGUGUCUUAGCUGGUUGUU(SEQ ID NO: 26) (G formerly at position 2 deleted)GGCAGUGUCUUAGCUGGUUGUU (SEQ ID NO: 27)(U formerly at position 1 deleted)

In embodiments where a nucleotide has been deleted relative to SEQ IDNO:2, it is contemplated that the designation of a modified nucleotidemay be adjusted accordingly.

In some embodiments, it is contemplated that an active strand having asequence that is at least 95% identical to SEQ ID NO:1 has amodification of a nucleotide at one or more of the following positions:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, or 22 with respect to either the 5′ or 3′ end of the strand. Inother embodiments, it is contemplated that an active strand may have thefollowing nucleotides modified: U at position 1 relative to SEQ ID NO:1;G at position 2 relative to SEQ ID NO:1; G at position 3 relative to SEQID NO:1; C at position 4 relative to SEQ ID NO:1; A at position 5relative to SEQ ID NO:1; G at position 6 relative to SEQ ID NO:1; U atposition 7 relative to SEQ ID NO:1; G at position 8 relative to SEQ IDNO:1; U at position 9 relative to SEQ ID NO:1; C at position 10 relativeto SEQ ID NO:1; U at position 11 relative to SEQ ID NO:1; U at position12 relative to SEQ ID NO:1; A at position 13 relative to SEQ ID NO:1; Gat position 14 relative to SEQ ID NO:1; C at position 15 relative to SEQID NO:1; U at position 16 relative to SEQ ID NO:1; G at position 17relative to SEQ ID NO:1; G at position 18 relative to SEQ ID NO:1; U atposition 19 relative to SEQ ID NO:1; U at position 20 relative to SEQ IDNO:1; G at position 21 relative to SEQ ID NO:1; and/or, U at position 22relative to SEQ ID NO:1. This means that the active strand may no longerhave the nucleotide at that position, but in the context of the sequenceof SEQ ID NO:1, the particular nucleotide in the active strand ismodified. This means its position may be altered by ±1 or ±2; forexample, the U at position 7 relative to SEQ ID NO:1, may be at position8 or at position 9 in the active strand because there has been aninsertion that affects its position number.

In some embodiments, it is contemplated that an active strand having asequence that is at least 95% identical to SEQ ID NO:2 has amodification of a nucleotide at one or more of the following positions:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, or 23 with respect to either the 5′ or 3′ end of the strand. Inother embodiments, it is contemplated that an active strand may have thefollowing nucleotides modified: U at position 1 relative to SEQ ID NO:2;G at position 2 relative to SEQ ID NO:2; G at position 3 relative to SEQID NO:2; C at position 4 relative to SEQ ID NO:2; A at position 5relative to SEQ ID NO:2; G at position 6 relative to SEQ ID NO:2; U atposition 7 relative to SEQ ID NO:2; G at position 8 relative to SEQ IDNO:2; U at position 9 relative to SEQ ID NO:2; C at position 10 relativeto SEQ ID NO:2; U at position 11 relative to SEQ ID NO:2; U at position12 relative to SEQ ID NO:2; A at position 13 relative to SEQ ID NO:2; Gat position 14 relative to SEQ ID NO:2; C at position 15 relative to SEQID NO:2; U at position 16 relative to SEQ ID NO:2; G at position 17relative to SEQ ID NO:2; G at position 18 relative to SEQ ID NO:2; U atposition 19 relative to SEQ ID NO:2; U at position 20 relative to SEQ IDNO:2; G at position 21 relative to SEQ ID NO:2; U at position 22relative to SEQ ID NO:2; and/or, U at position 23 relative to SEQ IDNO:2. This means that the active strand may no longer have thenucleotide at that position, but in the context of the sequence of SEQID NO:2, the particular nucleotide in the active strand is modified.This means its position may be altered by ±1 or ±2; for example, the Cat position 10 relative to SEQ ID NO:2, may be at position 8 or atposition 12 in the active strand because there has been an deletion orinsertion (respectively) that affects its position number.

In some embodiments, an active strand is 95% identical to SEQ ID NO:1(5′-UGGCAGUGUCUUAGCUGGUUGU-3′). In certain embodiments such an activestrand has the following sequence from 5′ to 3′ in which one nucleotideis substituted with a different ribonucleotide (A, C, G, or U), asrepresented by N:

NGGCAGUGUCUUAGCUGGUUGU (SEQ ID NO: 27) UNGCAGUGUCUUAGCUGGUUGU(SEQ ID NO: 28) UGNCAGUGUCUUAGCUGGUUGU (SEQ ID NO: 29)UGGNAGUGUCUUAGCUGGUUGU (SEQ ID NO: 30) UGGCNGUGUCUUAGCUGGUUGU(SEQ ID NO: 31) UGGCANUGUCUUAGCUGGUUGU (SEQ ID NO: 32)UGGCAGNGUCUUAGCUGGUUGU (SEQ ID NO: 33) UGGCAGUNUCUUAGCUGGUUGU(SEQ ID NO: 34) UGGCAGUGNCUUAGCUGGUUGU (SEQ ID NO: 35)UGGCAGUGUNUUAGCUGGUUGU (SEQ ID NO: 36) UGGCAGUGUCNUAGCUGGUUGU(SEQ ID NO: 37) UGGCAGUGUCUNAGCUGGUUGU (SEQ ID NO: 38)UGGCAGUGUCUUNGCUGGUUGU (SEQ ID NO: 39) UGGCAGUGUCUUANCUGGUUGU(SEQ ID NO: 40) UGGCAGUGUCUUAGNUGGUUGU (SEQ ID NO: 41)UGGCAGUGUCUUAGCNGGUUGU (SEQ ID NO: 42) UGGCAGUGUCUUAGCUNGUUGU(SEQ ID NO: 43) UGGCAGUGUCUUAGCUGNUUGU (SEQ ID NO: 44)UGGCAGUGUCUUAGCUGGNUGU (SEQ ID NO: 45) UGGCAGUGUCUUAGCUGGUNGU(SEQ ID NO: 46) UGGCAGUGUCUUAGCUGGUUNU (SEQ ID NO: 47)UGGCAGUGUCUUAGCUGGUUGN (SEQ ID NO: 48)

In some embodiments, an active strand is at least 95% identical to SEQID NO:2 (5′-UGGCAGUGUCUUAGCUGGUUGUU-3′), with a substitution of anucleotide relative to SEQ ID NO:2. In such embodiments, the sequence ofthe active strand includes one of the sequences disclosed above exceptit has an added U at the 3′ end or there is a substitution for the lastU in SEQ ID NO:2. In other embodiments, there is an active strand thatis at least 90% identical to SEQ ID NO:2 except there is a substitutionof two nucleotides relative to SEQ ID NO:2. In such embodiments, thereis an additional substitution in a sequence disclosed above except thereis an added U or there is a substitution for the last U in SEQ ID NO:2.

In some embodiments, an active strand is 95-100% identical to SEQ IDNO:1, which should include the sequences disclosed above. Other examplesof such active strands include active strands with an insertion of asingle nucleotide, as discussed below, in which the following sequencesfrom 5′ to 3′ have an insertion of a nucleotide designated as N, whichmay be an A, C, G, or U:

NUGGCAGUGUCUUAGCUGGUUGU (SEQ ID NO: 49) UNGGCAGUGUCUUAGCUGGUUGU(SEQ ID NO: 50) UGNGCAGUGUCUUAGCUGGUUGU (SEQ ID NO: 51)UGGNCAGUGUCUUAGCUGGUUGU (SEQ ID NO: 52) UGGCNAGUGUCUUAGCUGGUUGU(SEQ ID NO: 53) UGGCANGUGUCUUAGCUGGUUGU (SEQ ID NO: 54)UGGCAGNUGUCUUAGCUGGUUGU (SEQ ID NO: 55) UGGCAGUNGUCUUAGCUGGUUGU(SEQ ID NO: 56) UGGCAGUGNUCUUAGCUGGUUGU (SEQ ID NO: 57)UGGCAGUGUNCUUAGCUGGUUGU (SEQ ID NO: 58) UGGCAGUGUCNUUAGCUGGUUGU(SEQ ID NO: 59) UGGCAGUGUCUNUAGCUGGUUGU (SEQ ID NO: 60)UGGCAGUGUCUUNAGCUGGUUGU (SEQ ID NO: 61) UGGCAGUGUCUUANGCUGGUUGU(SEQ ID NO: 62) UGGCAGUGUCUUAGNCUGGUUGU (SEQ ID NO: 63)UGGCAGUGUCUUAGCNUGGUUGU (SEQ ID NO: 64) UGGCAGUGUCUUAGCUNGGUUGU(SEQ ID NO: 65) UGGCAGUGUCUUAGCUGNGUUGU (SEQ ID NO: 66)UGGCAGUGUCUUAGCUGGNUUGU (SEQ ID NO: 67) UGGCAGUGUCUUAGCUGGUNUGU(SEQ ID NO: 68) UGGCAGUGUCUUAGCUGGUUNGU (SEQ ID NO: 69)UGGCAGUGUCUUAGCUGGUUGNU (SEQ ID NO: 70) UGGCAGUGUCUUAGCUGGUUGUN(SEQ ID NO: 71)

In some embodiments, in addition to the single insertion shown above,there is a second insertion or addition elsewhere in the sequencerelative to SEQ ID NO:1. It is contemplated that the second insertionmay be after the nucleotide newly or previously located at position 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, or 23. Furthermore, in some embodiments, an active strand is 95%identical to SEQ ID NO:2 (5′-UGGCAGUGUCUUAGCUGGUUGUU-3′), which includesan insertion of a nucleotide relative to SEQ ID NO:2. In suchembodiments, the sequence of the active strand includes one of thesequences disclosed above except it has an added U at the 3′ end (exceptwhere there is an insertion at the 3′ end with respect to the added U).Embodiments include those with one or two insertions relative to the SEQID NO:2.

In some embodiments, an active strand is 95-100% identical to SEQ IDNO:2, which should include the sequences disclosed above. Such activestrands would be included in RNA molecules that can serve as a miR-34amimic. Other examples of such active strands include active strands withan insertion of a single nucleotide into the sequence of SEQ ID NO:2.

In certain embodiments, the active strand has a sequence that is or isat least 95% identical to SEQ ID NO:1 or SEQ ID NO:2. SEQ ID NO:1 (22nucleotides in length) is 95.7% identical to SEQ ID NO:2 (23 nucleotidesin length), and a fragment of 22 contiguous nucleotides in SEQ ID NO:2is 100% identical to SEQ ID:1.

It is noted that in some embodiments the sequence of the active strandconsists of SEQ ID NO:1, which means the active strand has a sequencethat is 100% identical to SEQ ID NO:1. In other embodiments, thesequence of the active strand consists of SEQ ID NO:2, which means theactive strand has a sequence that is 100% identical to SEQ ID NO:2. Inany of these embodiments, it is contemplated that an active strand mayinclude a modification of a nucleotide located at position 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 171, 18, 19, 20, 21, 22and/or 23 (where position 1 is the 5′ end of the strand) with respect tothe 5′ end of the active strand. This means the nucleotide at therecited position is modified, and this designation is independent of theidentity of the particular nucleotide at that recited position. Thisdesignation is position-based, as opposed to nucleotide-based. In otherembodiments, the designations are nucleotide-based. In certainembodiments, a designation may be position based with respect to the 3′end of the active strand; in such a case, the active strand may includea modification of a nucleotide located 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 171, 18, 19, 20, 21, 22 and/or 23 nucleotidesaway from the 3′ end of the active strand. In some embodiments,nucleotide-based designations set forth that an active strand may bemodified at the following nucleotides: U at position 1 in SEQ ID NO:2; Uat position 1 in SEQ ID NO:1 and at position 2 in SEQ ID NO:2; A atposition 2 in SEQ ID NO:1 and at position 3 in SEQ ID NO:2; A atposition 3 in SEQ ID NO:1 and at position 4 in SEQ ID NO:2; G atposition 4 in SEQ ID NO:1 and at position 5 in SEQ ID NO:2; G atposition 0.5 in SEQ ID NO:1 and at position 6 in SEQ ID NO:2; C atposition 6 in SEQ ID NO:1 and at position 7 in SEQ ID NO:2; A atposition 7 in SEQ ID NO:1 and at position 8 in SEQ ID NO:2; C atposition 8 in SEQ ID NO:1 and at position 9 in SEQ ID NO:2; G atposition 9 in SEQ ID NO:1 and at position 10 in SEQ ID NO:2; C atposition 10 in SEQ ID NO:1 and at position 11 in SEQ ID NO:2; G atposition 11 in SEQ ID NO:1 and at position 12 in SEQ ID NO:2; G atposition 12 in SEQ ID NO:1 and at position 13 in SEQ ID NO:2; U atposition 13 in SEQ ID NO:1 and at position 14 in SEQ ID NO:2; G atposition 14 in SEQ ID NO:1 and at position 15 in SEQ ID NO:2; A atposition 15 in SEQ ID NO:1 and at position 16 in SEQ ID NO:2; A atposition 16 in SEQ ID NO:1 and at position 17 in SEQ ID NO:2; U atposition 17 in SEQ ID NO:1 and at position 18 in SEQ ID NO:2; G atposition 18 in SEQ ID NO:1 and at position 19 in SEQ ID NO:2; C atposition 19 in SEQ ID NO:1 and at position 20 in SEQ ID NO:2; C atposition 20 in SEQ ID NO:1 and at position 21 in SEQ ID NO:2; and/or Aat position 22 in SEQ ID NO:2.

In embodiments concerning an miRNA-34c mimic, it is contemplated that anRNA molecule may contain an active strand that is or is at least 90, 91,92, 93, 94, 95, 96, 97, 98, 99 or 100% identical, or any range derivabletherein, to SEQ ID NO:5.

In some embodiments, an active strand is 95% identical to SEQ ID NO:5(5′-AGGCAGUGUAGUUAGCUGAUUGC-3′) (23-mer). In certain embodiments, theactive strand has the following sequence from 5′ to 3′ in which onenucleotide from SEQ ID NO:5 is deleted:

AGGCAGUGUAGUUAGCUGAUUG (SEQ ID NO: 72)(C formerly at position 23 deleted) AGGCAGUGUAGUUAGCUGAUUC(SEQ ID NO: 73) (G formerly at position 22 deleted)AGGCAGUGUAGUUAGCUGAUGC (SEQ ID NO: 74)(U formerly at position 21 deleted) AGGCAGUGUAGUUAGCUGAUGC(SEQ ID NO: 74) (U formerly at position 20 deleted)AGGCAGUGUAGUUAGCUGUUGC (SEQ ID NO: 75)(A formerly at position 19 deleted) AGGCAGUGUAGUUAGCUAUUGC(SEQ ID NO: 76) (G formerly at position 18 deleted)AGGCAGUGUAGUUAGCGAUUGC (SEQ ID NO: 77)(U formerly at position 17 deleted) AGGCAGUGUAGUUAGUGAUUGC(SEQ ID NO: 78) (C formerly at position 16 deleted)AGGCAGUGUAGUUACUGAUUGC (SEQ ID NO: 79)(G formerly at position 15 deleted) AGGCAGUGUAGUUGCUGAUUGC(SEQ ID NO: 80) (A formerly at position 14 deleted)AGGCAGUGUAGUAGCUGAUUGC (SEQ ID NO: 81)(U formerly at position 13 deleted) AGGCAGUGUAGUAGCUGAUUGC(SEQ ID NO: 81) (U formerly at position 12 deleted)AGGCAGUGUAUUAGCUGAUUGC (SEQ ID NO: 82)(G formerly at position 11 deleted) AGGCAGUGUGUUAGCUGAUUGC(SEQ ID NO: 83) (A formerly at position 10 deleted)AGGCAGUGAGUUAGCUGAUUGC (SEQ ID NO: 84)(U formerly at position 9 deleted) AGGCAGUUAGUUAGCUGAUUGC(SEQ ID NO: 85) (G formerly at position 8 deleted)AGGCAGGUAGUUAGCUGAUUGC (SEQ ID NO: 86)(U formerly at position 7 deleted) AGGCAUGUAGUUAGCUGAUUGC(SEQ ID NO: 87) (G formerly at position 6 deleted)AGGCGUGUAGUUAGCUGAUUGC (SEQ ID NO: 88)(A formerly at position 5 deleted) AGGAGUGUAGUUAGCUGAUUGC(SEQ ID NO: 89) (C formerly at position 4 deleted)AGCAGUGUAGUUAGCUGAUUGC (SEQ ID NO: 90)(G formerly at position 3 deleted) AGCAGUGUAGUUAGCUGAUUGC(SEQ ID NO: 90) (G formerly at position 2 deleted)GGCAGUGUAGUUAGCUGAUUGC (SEQ ID NO: 91)(A formerly at position 1 deleted)

In embodiments where a nucleotide has been deleted relative to SEQ IDNO:5, it is contemplated that the designation of a modified nucleotidemay be adjusted accordingly.

In some embodiments, it is contemplated that an active strand having asequence that is at least 95% identical to SEQ ID NO:5 has amodification of a nucleotide at one or more of the following positions:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, or 23 with respect to either the 5′ or 3′ end of the strand. Inother embodiments, it is contemplated that an active strand may have thefollowing nucleotides modified: A at position 1 relative to SEQ ID NO:5;G at position 2 relative to SEQ ID NO:5; G at position 3 relative to SEQID NO:5; C at position 4 relative to SEQ ID NO:5; A at position 5relative to SEQ ID NO:5; G at position 6 relative to SEQ ID NO:5; U atposition 7 relative to SEQ ID NO:5; G at position 8 relative to SEQ IDNO:5; U at position 9 relative to SEQ ID NO:5; A at position 10 relativeto SEQ ID NO:5; G at position 11 relative to SEQ ID NO:5; U at position12 relative to SEQ ID NO:5; U at position 13 relative to SEQ ID NO:5; Aat position 14 relative to SEQ ID NO:5; G at position 15 relative to SEQID NO:5; C at position 16 relative to SEQ ID NO:5; U at position 17relative to SEQ ID NO:5; G at position 18 relative to SEQ ID NO:5; A atposition 19 relative to SEQ ID NO:5; U at position 20 relative to SEQ IDNO:5; U at position 21 relative to SEQ ID NO:5; G at position 22relative to SEQ ID NO:5; and/or C at position 23 relative to SEQ IDNO:5. This means that the active strand may no longer have thenucleotide at that position, but in the context of the sequence of SEQID NO:5, the particular nucleotide in the active strand is modified.This means its position may be altered by +1 or +2; for example, the Uat position 7 relative to SEQ ID NO:5, may be at position 7 or atposition 8 in the active strand because there has been a deletion or aninsertion, respectively, that affects its position number.

In some embodiments, an active strand is at least 95% identical to SEQID NO:5 (5′-AGGCAGUGUAGUUAGCUGAUUGC-3′). In certain embodiments such anactive strand has the following sequence from 5′ to 3′ in which onenucleotide is substituted with a different ribonucleotide (A, C, G, orU), as represented by N:

NGGCAGUGUAGUUAGCUGAUUGC (SEQ ID NO: 92) ANGCAGUGUAGUUAGCUGAUUGC(SEQ ID NO: 93) AGNCAGUGUAGUUAGCUGAUUGC (SEQ ID NO: 94)AGGNAGUGUAGUUAGCUGAUUGC (SEQ ID NO: 95) AGGCNGUGUAGUUAGCUGAUUGC(SEQ ID NO: 96) AGGCANUGUAGUUAGCUGAUUGC (SEQ ID NO: 97)AGGCAGNGUAGUUAGCUGAUUGC (SEQ ID NO: 98) AGGCAGUNUAGUUAGCUGAUUGC(SEQ ID NO: 99) AGGCAGUGNAGUUAGCUGAUUGC (SEQ ID NO: 100)AGGCAGUGUNGUUAGCUGAUUGC (SEQ ID NO: 101) AGGCAGUGUANUUAGCUGAUUGC(SEQ ID NO: 102) AGGCAGUGUAGNUAGCUGAUUGC (SEQ ID NO: 103)AGGCAGUGUAGUNAGCUGAUUGC (SEQ ID NO: 104) AGGCAGUGUAGUUNGCUGAUUGC(SEQ ID NO: 105) AGGCAGUGUAGUUANCUGAUUGC (SEQ ID NO: 106)AGGCAGUGUAGUUAGNUGAUUGC (SEQ ID NO: 107) AGGCAGUGUAGUUAGCNGAUUGC(SEQ ID NO: 108) AGGCAGUGUAGUUAGCUNAUUGC (SEQ ID NO: 109)AGGCAGUGUAGUUAGCUGNUUGC (SEQ ID NO: 110) AGGCAGUGUAGUUAGCUGANUGC(SEQ ID NO: 111) AGGCAGUGUAGUUAGCUGAUNGC (SEQ ID NO: 112)AGGCAGUGUAGUUAGCUGAUUNC (SEQ ID NO: 113) AGGCAGUGUAGUUAGCUGAUUGN(SEQ ID NO: 114)

In some embodiments, an active strand is at least 90% identical to SEQID NO:5. In such embodiments, the sequence of the active strand includesone of the sequences disclosed above and there may be one otherinsertion, deletion, or substitution (such as those discussed herein) inaddition to the substitution shown above.

In some embodiments, an active strand is 95-100% identical to SEQ IDNO:5. Examples of such active strands include active strands with aninsertion of a single nucleotide, as discussed below, in which thefollowing sequences from 5′ to 3′ have an insertion of a nucleotidedesignated as N, which may be an A, C, G, or U:

NAGGCAGUGUAGUUAGCUGAUUGC (SEQ ID NO: 115) ANGGCAGUGUAGUUAGCUGAUUGC(SEQ ID NO: 116) AGNGCAGUGUAGUUAGCUGAUUGC (SEQ ID NO: 117)AGGNCAGUGUAGUUAGCUGAUUGC (SEQ ID NO: 118) AGGCNAGUGUAGUUAGCUGAUUGC(SEQ ID NO: 119) AGGCANGUGUAGUUAGCUGAUUGC (SEQ ID NO: 120)AGGCAGNUGUAGUUAGCUGAUUGC (SEQ ID NO: 121) AGGCAGUNGUAGUUAGCUGAUUGC(SEQ ID NO: 122) AGGCAGUGNUAGUUAGCUGAUUGC (SEQ ID NO: 123)AGGCAGUGUNAGUUAGCUGAUUGC (SEQ ID NO: 124) AGGCAGUGUANGUUAGCUGAUUGC(SEQ ID NO: 125) AGGCAGUGUAGNUUAGCUGAUUGC (SEQ ID NO: 126)AGGCAGUGUAGUNUAGCUGAUUGC (SEQ ID NO: 127) AGGCAGUGUAGUUNAGCUGAUUGC(SEQ ID NO: 128) GGCAGUGUAGUUANGCUGAUUGC (SEQ ID NO: 129)AGGCAGUGUAGUUAGNCUGAUUGC (SEQ ID NO: 130) AGGCAGUGUAGUUAGCNUGAUUGC(SEQ ID NO: 131) AGGCAGUGUAGUUAGCUNGAUUGC (SEQ ID NO: 132)AGGCAGUGUAGUUAGCUGNAUUGC (SEQ ID NO: 133) AGGCAGUGUAGUUAGCUGANUUGC(SEQ ID NO: 134) AGGCAGUGUAGUUAGCUGAUNUGC (SEQ ID NO: 135)AGGCAGUGUAGUUAGCUGAUUNGC (SEQ ID NO: 136) AGGCAGUGUAGUUAGCUGAUUGNC(SEQ ID NO: 137) AGGCAGUGUAGUUAGCUGAUUGCN (SEQ ID NO: 138)

In some embodiments, in addition to the single insertion shown above,there is a second insertion or addition elsewhere in the sequencerelative to SEQ ID NO:5. It is contemplated that the second insertionmay be after the nucleotide newly or previously located at position 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, or 23.

In certain embodiments involving a miR-34a mimic, the passenger strandhas a sequence that is or is at least 95% identical to SEQ ID NO:3 orSEQ ID NO:4. SEQ ID NO:3 (20 nucleotides in length) is 90.9% identicalto SEQ ID NO:4 (22 nucleotides in length), and a fragment of 20contiguous nucleotides in SEQ ID NO:4 is 100% identical to SEQ ID:3.

It is noted that in some embodiments the sequence of the passengerstrand consists of SEQ ID NO:3, which means the passenger strand has asequence that is 100% identical to SEQ ID NO:3. In other embodiments,the sequence of the passenger strand consists of SEQ ID NO:4, whichmeans the passenger strand has a sequence that is 100% identical to SEQID NO:4. In any of these embodiments, it is contemplated that anpassenger strand may include a modification of a nucleotide located atposition 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 171, 18,19, 20, 21, and/or 22 (where position 1 is the 5′ end of the strand)with respect to the 5′ end of the passenger strand. This means thenucleotide at the recited position is modified, and this designation isindependent of the identity of the particular nucleotide at that recitedposition. This designation is position-based, as opposed tonucleotide-based. In other embodiments, the designations arenucleotide-based. In certain embodiments, a designation may be positionbased with respect to the 3′ end of the passenger strand; in such acase, the passenger strand may include a modification of a nucleotidelocated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 171, 18,19, 20, 21, and/or 22 nucleotides away from the 3′ end of the passengerstrand.

It is contemplated that an RNA molecule may contain a passenger strandthat is or is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%identical, or any range derivable therein, to SEQ ID NO:3. In otherembodiments, a passenger strand is or is at least 90, 91, 92, 93, 94,95, 96, 97, 98, 99 or 100% identical, or any range derivable therein, toSEQ ID NO:4.

In some embodiments, a passenger strand is 95% identical to SEQ ID NO:3(5′-ACAACCAGCUAAGACACUGCCA-3′) (22-mer). In certain embodiments, theactive strand has the following sequence from 5′ to 3′ in which onenucleotide from SEQ ID NO:3 is deleted:

ACAACCAGCUAAGACACUGCC (SEQ ID NO: 139) (A formerly at position 22 deleted)ACAACCAGCUAAGACACUGCA (SEQ ID NO: 140) (C formerly at position 21 deleted)ACAACCAGCUAAGACACUGCA (SEQ ID NO: 140) (C formerly at position 20 deleted)ACAACCAGCUAAGACACUCCA (SEQ ID NO: 141) (G formerly at position 19 deleted)ACAACCAGCUAAGACACGCCA (SEQ ID NO: 142) (U formerly at position 18 deleted)ACAACCAGCUAAGACAUGCCA (SEQ ID NO: 143) (C formerly at position 17 deleted)ACAACCAGCUAAGACCUGCCA (SEQ ID NO: 144) (A formerly at position 16 deleted)ACAACCAGCUAAGAACUGCCA (SEQ ID NO: 145) (C formerly at position 15 deleted)ACAACCAGCUAAGCACUGCCA (SEQ ID NO: 146) (A formerly at position 14 deleted)ACAACCAGCUAAACACUGCCA (SEQ ID NO: 147) (G formerly at position 13 deleted)ACAACCAGCUAGACACUGCCA (SEQ ID NO: 148) (A formerly at position 12 deleted)ACAACCAGCUAGACACUGCCA (SEQ ID NO: 148) (A formerly at position 11 deleted)ACAACCAGCAAGACACUGCCA (SEQ ID NO: 149) (U formerly at position 10 deleted)ACAACCAGUAAGACACUGCCA (SEQ ID NO: 150) (C formerly at position 9 deleted)ACAACCACUAAGACACUGCCA (SEQ ID NO: 151) (G formerly at position 8 deleted)ACAACCGCUAAGACACUGCCA (SEQ ID NO: 152) (A formerly at position 7 deleted)ACAACAGCUAAGACACUGCCA (SEQ ID NO: 153) (C formerly at position 6 deleted)ACAACAGCUAAGACACUGCCA (SEQ ID NO: 153) (C formerly at position 5 deleted)ACACCAGCUAAGACACUGCCA (SEQ ID NO: 154) (A formerly at position 4 deleted)ACACCAGCUAAGACACUGCCA (SEQ ID NO: 154) (A formerly at position 3 deleted)AAACCAGCUAAGACACUGCCA (SEQ ID NO: 155) (C formerly at position 2 deleted)CAACCAGCUAAGACACUGCCA (SEQ ID NO: 156) (A formerly at position 1 deleted)

In embodiments where a nucleotide has been deleted relative to SEQ IDNO:3, it is contemplated that the designation of a modified nucleotidemay be adjusted accordingly.

In some embodiments, it is contemplated that an active strand having asequence that is at least 95% identical to SEQ ID NO:3 has amodification of a nucleotide at one or more of the following positions:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, or 22 with respect to either the 5′ or 3′ end of the strand: A atposition 1 relative to SEQ ID NO:3; C at position 2 relative to SEQ IDNO:3; A at position 3 relative to SEQ ID NO:3; A at position 4 relativeto SEQ ID NO:3; C at position 5 relative to SEQ ID NO:3; C at position 6relative to SEQ ID NO:3; A at position 7 relative to SEQ ID NO:3; G atposition 8 relative to SEQ ID NO:3; C at position 9 relative to SEQ IDNO:3; U at position 10 relative to SEQ ID NO:3; A at position 11relative to SEQ ID NO:3; A at position 12 relative to SEQ ID NO:3; G atposition 13 relative to SEQ ID NO:3; A at position 14 relative to SEQ IDNO:3; C at position 15 relative to SEQ ID NO:3; A at position 16relative to SEQ ID NO:3; C at position 17 relative to SEQ ID NO:3; U atposition 18 relative to SEQ ID NO:3; G at position 19 relative to SEQ IDNO:3; C at position 20 relative to SEQ ID NO:3; C at position 21relative to SEQ ID NO:3; and/or A at position 22 relative to SEQ IDNO:3. This means that the passenger strand may no longer have thenucleotide at that position, but in the context of the sequence of SEQID NO:3, the particular nucleotide in the active strand is modified.This means its position may be altered by −1 or +1; for example, the Gat position 11 relative to SEQ ID NO:3, may be at position 10 or atposition 12 in a passenger strand because there has been an insertion ordeletion that affects its position number.

In some embodiments, a passenger strand is 95% identical to SEQ ID NO:4(5′-AACAACCAGCUAAGACACUGCCA-3′), which is 2 bases longer than SEQ IDNO:2. In certain embodiments, the passenger strand has the followingsequence from 5′ to 3′ in which one nucleotide from SEQ ID NO:4 isdeleted:

AACAACCAGCUAAGACACUGCC (SEQ ID NO: 157) (A formerly at position 23 deleted)AACAACCAGCUAAGACACUGCA (SEQ ID NO: 158) (C formerly at position 22 deleted)AACAACCAGCUAAGACACUGCA (SEQ ID NO: 158) (C formerly at position 21 deleted)AACAACCAGCUAAGACACUCCA (SEQ ID NO: 159) (G formerly at position 20 deleted)AACAACCAGCUAAGACACGCCA (SEQ ID NO: 160) (U formerly at position 19 deleted)AACAACCAGCUAAGACAUGCCA (SEQ ID NO: 161) (C formerly at position 18 deleted)AACAACCAGCUAAGACCUGCCA (SEQ ID NO: 162) (A formerly at position 17 deleted)AACAACCAGCUAAGAACUGCCA (SEQ ID NO: 163) (C formerly at position 16 deleted)AACAACCAGCUAAGCACUGCCA (SEQ ID NO: 164) (A formerly at position 15 deleted)AACAACCAGCUAAACACUGCCA (SEQ ID NO: 165) (G formerly at position 14 deleted)AACAACCAGCUAGACACUGCCA (SEQ ID NO: 166) (A formerly at position 13 deleted)AACAACCAGCUAGACACUGCCA (SEQ ID NO: 166) (A formerly at position 12 deleted)AACAACCAGCAAGACACUGCCA (SEQ ID NO: 167) (U formerly at position 11 deleted)AACAACCAGUAAGACACUGCCA (SEQ ID NO: 168) (C formerly at position 10 deleted)AACAACCACUAAGACACUGCCA (SEQ ID NO: 169) (G formerly at position 9 deleted)AACAACCGCUAAGACACUGCCA (SEQ ID NO: 170) (A formerly at position 8 deleted)AACAACAGCUAAGACACUGCCA (SEQ ID NO: 171) (C formerly at position 7 deleted)AACAACAGCUAAGACACUGCCA (SEQ ID NO: 171) (C formerly at position 6 deleted)AACACCAGCUAAGACACUGCCA (SEQ ID NO: 172) (A formerly at position 5 deleted)AACACCAGCUAAGACACUGCCA (SEQ ID NO: 173) (A formerly at position 4 deleted)AAAACCAGCUAAGACACUGCCA (SEQ ID NO: 174) (C formerly at position 3 deleted)ACAACCAGCUAAGACACUGCCA (SEQ ID NO: 175) (A formerly at position 2 deleted)ACAACCAGCUAAGACACUGCCA (SEQ ID NO: 175) (A formerly at position 1 deleted)

In embodiments where a nucleotide has been deleted relative to SEQ IDNO:4, it is contemplated that the designation of a modified nucleotidemay be adjusted accordingly. In some embodiments, it is contemplatedthat an active strand having a sequence that is at least 95% identicalto SEQ ID NO:4 has a modification of a nucleotide at one or more of thefollowing positions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21 or 22 with respect to either the 5′ or 3′ end ofthe strand.

In other embodiments, it is contemplated that an active strand may havethe following nucleotides modified: A at position 1 relative to SEQ IDNO:4; A at position 2 relative to SEQ ID NO:4; C at position 3 relativeto SEQ ID NO:4; A at position 4 relative to SEQ ID NO:4; A at position 5relative to SEQ ID NO:4; C at position 6 relative to SEQ ID NO:4; C atposition 7 relative to SEQ ID NO:4; A at position 8 relative to SEQ IDNO:4; G at position 9 relative to SEQ ID NO:4; C at position 10 relativeto SEQ ID NO:4; U at position 11 relative to SEQ ID NO:4; A at position12 relative to SEQ ID NO:4; A at position 13 relative to SEQ ID NO:4; Gat position 14 relative to SEQ ID NO:4; A at position 15 relative to SEQID NO:4; C at position 16 relative to SEQ ID NO:4; A at position 17relative to SEQ ID NO:4; C at position 18 relative to SEQ ID NO:4; U atposition 19 relative to SEQ ID NO:4; G at position 20 relative to SEQ IDNO:4; C at position 21 relative to SEQ ID NO:4; C at position 23relative to SEQ ID NO:4; and/or A at position 23 relative to SEQ IDNO:4. This means that the active strand may no longer have thenucleotide at that position, but in the context of the sequence of SEQID NO:2, the particular nucleotide in the active strand is modified.This means its position may be altered by −1 or −2; for example, the Cat position 11 relative to SEQ ID NO:2, may be at position 10 in theactive strand because there has been a deletion that affects itsposition number.

In some embodiments, a passenger strand is 95% identical to SEQ ID NO:3(5′-ACAACCAGCUAAGACACUGCCA-3′). In certain embodiments such a passengerstrand has the following sequence from 5′ to 3′ in which one nucleotideis substituted with a different ribonucleotide (A, C, G, or U), asrepresented by N:

NCAACCAGCUAAGACACUGCCA (SEQ ID NO: 176) ANAACCAGCUAAGACACUGCCA(SEQ ID NO: 177) ACNACCAGCUAAGACACUGCCA (SEQ ID NO: 178)ACANCCAGCUAAGACACUGCCA (SEQ ID NO: 179) ACAANCAGCUAAGACACUGCCA(SEQ ID NO: 180) ACAACNAGCUAAGACACUGCCA (SEQ ID NO: 181)ACAACCNGCUAAGACACUGCCA (SEQ ID NO: 182) ACAACCANCUAAGACACUGCCA(SEQ ID NO: 183) ACAACCAGNUAAGACACUGCCA (SEQ ID NO: 184)ACAACCAGCNAAGACACUGCCA (SEQ ID NO: 185) ACAACCAGCUNAGACACUGCCA(SEQ ID NO: 186) ACAACCAGCUANGACACUGCCA (SEQ ID NO: 187)ACAACCAGCUAANACACUGCCA (SEQ ID NO: 188) ACAACCAGCUAAGNCACUGCCA(SEQ ID NO: 189) ACAACCAGCUAAGANACUGCCA (SEQ ID NO: 190)ACAACCAGCUAAGACNCUGCCA (SEQ ID NO: 191) ACAACCAGCUAAGACANUGCCA(SEQ ID NO: 192) ACAACCAGCUAAGACACNGCCA (SEQ ID NO: 193)ACAACCAGCUAAGACACUNCCA (SEQ ID NO: 194) ACAACCAGCUAAGACACUGNCA(SEQ ID NO: 195) ACAACCAGCUAAGACACUGCNA (SEQ ID NO: 196)ACAACCAGCUAAGACACUGCCN (SEQ ID NO: 197)

In some embodiments, in addition to the single substitution shown above,there is a second substitution elsewhere in the sequence relative to SEQID NO:3. It is further contemplated that there may be a secondsubstitution with one of the substitutions described in a passengerstrand described above, or one or two deletions of nucleotides inaddition to the substitution described above.

In some embodiments, a passenger strand is 95-100% identical to SEQ IDNO:3, which should include the sequences disclosed above. Other examplesof such passenger strands include passenger strands with an insertion ofa single nucleotide, as discussed below, in which the followingsequences from 5′ to 3′ have an insertion of a nucleotide designated asN, which may be an A, C, G, or U:

NACAACCAGCUAAGACACUGCCA (SEQ ID NO: 198) ANCAACCAGCUAAGACACUGCCA(SEQ ID NO: 199) ACNAACCAGCUAAGACACUGCCA (SEQ ID NO: 200)ACANACCAGCUAAGACACUGCCA (SEQ ID NO: 201) ACAANCCAGCUAAGACACUGCCA(SEQ ID NO: 202) ACAACNCAGCUAAGACACUGCCA (SEQ ID NO: 203)ACAACCNAGCUAAGACACUGCCA (SEQ ID NO: 204) ACAACCANGCUAAGACACUGCCA(SEQ ID NO: 205) ACAACCAGNCUAAGACACUGCCA (SEQ ID NO: 206)ACAACCAGCNUAAGACACUGCCA (SEQ ID NO: 207) ACAACCAGCUNAAGACACUGCCA(SEQ ID NO: 208) ACAACCAGCUANAGACACUGCCA (SEQ ID NO: 209)ACAACCAGCUAANGACACUGCCA (SEQ ID NO: 210) ACAACCAGCUAAGNACACUGCCA(SEQ ID NO: 211) ACAACCAGCUAAGANCACUGCCA (SEQ ID NO: 212)ACAACCAGCUAAGACNACUGCCA (SEQ ID NO: 213) ACAACCAGCUAAGACANCUGCCA(SEQ ID NO: 214) ACAACCAGCUAAGACACNUGCCA (SEQ ID NO: 215)ACAACCAGCUAAGACACUNGCCA (SEQ ID NO: 216) ACAACCAGCUAAGACACUGNCCA(SEQ ID NO: 217) ACAACCAGCUAAGACACUGCNCA (SEQ ID NO: 218)ACAACCAGCUAAGACACUGCCNA (SEQ ID NO: 219) ACAACCAGCUAAGACACUGCCAN(SEQ ID NO: 220)

In some embodiments, in addition to the insertion in the passengerstrand shown above relative to SEQ ID NO:3, there may be a secondinsertion elsewhere in the strand. Combinations of insertions in thepassengers strands shown above are also contemplated for additionalpassenger strands.

In some embodiments, a passenger strand is or is at least 90% identicalto SEQ ID NO:4 (5′-AACAACCAGCUAAGACACUGCCA-3′). In certain embodiments,such a passenger strand has the following sequence from 5′ to 3′ inwhich one or two nucleotides is substituted with a differentribonucleotide. In certain embodiment there is one substitution asrepresented by N:

NAACAACCAGCUAAGACACUGCCA (SEQ ID NO: 221) ANACAACCAGCUAAGACACUGCCA(SEQ ID NO: 222) AANCAACCAGCUAAGACACUGCCA (SEQ ID NO: 223)AACNAACCAGCUAAGACACUGCCA (SEQ ID NO: 224) AACANACCAGCUAAGACACUGCCA(SEQ ID NO: 225) AACAANCCAGCUAAGACACUGCCA (SEQ ID NO: 226)AACAACNCAGCUAAGACACUGCCA (SEQ ID NO: 227) AACAACCNAGCUAAGACACUGCCA(SEQ ID NO: 228) AACAACCANGCUAAGACACUGCCA (SEQ ID NO: 229)AACAACCAGNCUAAGACACUGCCA (SEQ ID NO: 230) AACAACCAGCNUAAGACACUGCCA(SEQ ID NO: 231) AACAACCAGCUNAAGACACUGCCA (SEQ ID NO: 232)AACAACCAGCUANAGACACUGCCA (SEQ ID NO: 233) AACAACCAGCUAANGACACUGCCA(SEQ ID NO: 234) AACAACCAGCUAAGNACACUGCCA (SEQ ID NO: 235)AACAACCAGCUAAGANCACUGCCA (SEQ ID NO: 236) AACAACCAGCUAAGACNACUGCCA(SEQ ID NO: 237) AACAACCAGCUAAGACANCUGCCA (SEQ ID NO: 238)AACAACCAGCUAAGACACNUGCCA (SEQ ID NO: 239) AACAACCAGCUAAGACACUNGCCA(SEQ ID NO: 240) AACAACCAGCUAAGACACUGNCCA (SEQ ID NO: 241)AACAACCAGCUAAGACACUGCNCA (SEQ ID NO: 242) AACAACCAGCUAAGACACUGCCNA(SEQ ID NO: 243) AACAACCAGCUAAGACACUGCCAN (SEQ ID NO: 244)

In some embodiments, in addition to the single substitution shown above,there is a second substitution elsewhere in the sequence relative to SEQID NO:4. Moreover, any combination of substitutions shown above in thepassenger strands is contemplated.

In some embodiments, a passenger strand is 95-100% identical to SEQ IDNO:4, which should include the sequences disclosed above. Other examplesof such passenger strands include passenger strands with an insertion ofa single nucleotide into the sequence of SEQ ID NO:4.

It is noted that in some embodiments the sequence of the passengerstrand consists of SEQ ID NO:3, which means the passenger strand has asequence that is 100% identical to SEQ ID NO:3. In other embodiments,the sequence of the passenger strand consists of SEQ ID NO:4, whichmeans the passenger strand has a sequence that is 100% identical to SEQID NO:4. In any of these embodiments, it is contemplated that apassenger strand may include a modification of a nucleotide located atposition 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 171, 18,19, 20, 21, and/or 22 (where position 1 is the 5′ end of the strand)with respect to the 5′ end of the passenger strand. This means thenucleotide at the recited position is modified, and this designation isindependent of the identity of the particular nucleotide at that recitedposition. This designation is position-based, as opposed tonucleotide-based. In other embodiments, the designations arcnucleotide-based. In certain embodiments, a designation may be positionbased with respect to the 3′ end of the passenger strand; in such acase, the passenger strand may include a modification of a nucleotidelocated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 171, 18,19, 20, 21, and/or 22 nucleotides away from the 3′ end of the passengerstrand.

Any embodiments discussed herein in which a modified nucleotide wasidentified as nucleotide-based may be implemented in other embodiments amodified nucleotide that is position-based using the position of theidentified nucleotide. This applies to active strands, as well aspassenger strands.

In some embodiments, nucleotide-based designations set forth that apassenger strand may be modified at the following nucleotides: A atposition 1 in SEQ ID NO:4; A at position 2 in SEQ ID NO:4; C at position3 in SEQ ID NO:4; A at position 4 in SEQ ID NO:4; A at position 5 in SEQID NO:4; C at position 6 in SEQ ID NO:4; C at position 7 in SEQ ID NO:4;A at position 8 in SEQ ID NO:4; G at position 9 in SEQ ID NO:4; C atposition 10 in SEQ ID NO:4; U at position 11 in SEQ ID NO:4; A atposition 12 in SEQ ID NO:4; A at position 13 in SEQ ID NO:4; G atposition 14 in SEQ ID NO:4; A at position 15 in SEQ ID NO:4; C atposition 16 in SEQ ID NO:4; A at position 17 in SEQ ID NO:4; C atposition 18 in SEQ ID NO:4; U at position 19 in SEQ ID NO:4; G atposition 20 in SEQ ID NO:4; C at position 21 in SEQ ID NO:4; C atposition 22 in SEQ ID NO:4; and/or A at position 23 in SEQ ID NO:4.

In certain embodiments involving a miR-34c mimic, the passenger strandhas a sequence that is or is at least 95% identical to SEQ ID NO:6.

It is noted that in some embodiments the sequence of the passengerstrand consists of SEQ ID NO:6, which means the passenger strand has asequence that is 100% identical to SEQ ID NO:6. In any of theseembodiments, it is contemplated that an passenger strand may include amodification of a nucleotide located at position 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 171, 18, 19, 20, 21, 22 and/or 23 (whereposition 1 is the 5′ end of the strand) with respect to the 5′ end ofthe passenger strand. This means the nucleotide at the recited positionis modified, and this designation is independent of the identity of theparticular nucleotide at that recited position. This designation isposition-based, as opposed to nucleotide-based. In other embodiments,the designations are nucleotide-based. In certain embodiments, adesignation may be position based with respect to the 3′ end of thepassenger strand; in such a case, the passenger strand may include amodification of a nucleotide located 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 171, 18, 19, 20, 21, and/or 22 nucleotides away fromthe 3′ end of the passenger strand.

It is contemplated that an RNA molecule may contain a passenger strandthat is or is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%identical, or any range derivable therein, to SEQ ID NO:6.

In some embodiments, a passenger strand is 95% identical to SEQ ID NO:6(5′-GCAAUCAGCUAACUACACUGCCU-3′) (23-mer). In certain embodiments, thepassenger strand has the following sequence from 5′ to 3′ in which onenucleotide from SEQ ID NO:6 is deleted:

GCAAUCAGCUAACUACACUGCC (SEQ ID NO: 245) (U formerly at position 23 deleted)GCAAUCAGCUAACUACACUGCU (SEQ ID NO: 246) (C formerly at position 22 deleted)GCAAUCAGCUAACUACACUGCU (SEQ ID NO: 247) (C formerly at position 21 deleted)GCAAUCAGCUAACUACACUCCU (SEQ ID NO: 248) (G formerly at position 20 deleted)GCAAUCAGCUAACUACACGCCU (SEQ ID NO: 249) (U formerly at position 19 deleted)GCAAUCAGCUAACUACAUGCCU (SEQ ID NO: 250) (C formerly at position 18 deleted)GCAAUCAGCUAACUACCUGCCU (SEQ ID NO: 251) (A formerly at position 17 deleted)GCAAUCAGCUAACUAACUGCCU (SEQ ID NO: 252) (C formerly at position 16 deleted)GCAAUCAGCUAACUCACUGCCU (SEQ ID NO: 253) (A formerly at position 15 deleted)GCAAUCAGCUAACACACUGCCU (SEQ ID NO: 254) (U formerly at position 14 deleted)GCAAUCAGCUAAUACACUGCCU (SEQ ID NO: 255) (C formerly at position 13 deleted)GCAAUCAGCUACUACACUGCCU (SEQ ID NO: 256) (A formerly at position 12 deleted)GCAAUCAGCUACUACACUGCCU (SEQ ID NO: 257) (A formerly at position 11 deleted)GCAAUCAGCAACUACACUGCCU (SEQ ID NO: 258) (U formerly at position 10 deleted)GCAAUCAGUAACUACACUGCCU (SEQ ID NO: 259) (C formerly at position 9 deleted)GCAAUCACUAACUACACUGCCU (SEQ ID NO: 260) (G formerly at position 8 deleted)GCAAUCGCUAACUACACUGCCU (SEQ ID NO: 261) (A formerly at position 7 deleted)GCAAUAGCUAACUACACUGCCU (SEQ ID NO: 262) (C formerly at position 6 deleted)GCAACAGCUAACUACACUGCCU (SEQ ID NO: 263) (U formerly at position 5 deleted)GCAUCAGCUAACUACACUGCCU (SEQ ID NO: 264) (A formerly at position 4 deleted)GCAUCAGCUAACUACACUGCCU (SEQ ID NO: 265) (A formerly at position 3 deleted)GAAUCAGCUAACUACACUGCCU (SEQ ID NO: 266) (C formerly at position 2 deleted)CAAUCAGCUAACUACACUGCCU (SEQ ID NO: 267) (G formerly at position 1 deleted)

In embodiments where a nucleotide has been deleted relative to SEQ IDNO:6, it is contemplated that the designation of a modified nucleotidemay be adjusted accordingly. In some embodiments, it is contemplatedthat an active strand having a sequence that is at least 95% identicalto SEQ ID NO:6 has a modification of a nucleotide at one or more of thefollowing positions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, or 21 with respect to either the 5′ or 3′ end of thestrand: G at position 1 relative to SEQ ID NO:6; C at position 2relative to SEQ ID NO:6; A at position 3 relative to SEQ ID NO:6; A atposition 4 relative to SEQ ID NO:6; U at position 5 relative to SEQ IDNO:6; C at position 6 relative to SEQ ID NO:6; A at position 7 relativeto SEQ ID NO:6; G at position 8 relative to SEQ ID NO:6; C at position 9relative to SEQ ID NO:6; U at position 2 relative to SEQ ID NO:6; A atposition 11 relative to SEQ ID NO:6; A at position 12 relative to SEQ IDNO:6; C at position 13 relative to SEQ ID NO:6; U at position 14relative to SEQ ID NO:6; A at position 15 relative to SEQ ID NO:6; C atposition 16 relative to SEQ ID NO:6; A at position 17 relative to SEQ IDNO:6; C at position 18 relative to SEQ ID NO:6; U at position 19relative to SEQ ID NO:6; G at position 20 relative to SEQ ID NO:6; C atposition 21 relative to SEQ ID NO:6; C at position 22 relative to SEQ IDNO:6; and/or, U at position 23 relative to SEQ ID NO:6. In theseembodiments, this means that the passenger strand may no longer have thenucleotide at that position, but in the context of the sequence of SEQID NO:6, the particular nucleotide in the passenger strand is modified.This means its position may be altered by −1 or +1; for example, the Aat position 11 relative to SEQ ID NO:6, may be at position 10 or atposition 12 in a passenger strand because there has been an insertion ordeletion that affects its position number.

In some embodiments, a passenger strand is 95% identical to SEQ ID NO:6(5′-GCAAUCAGCUAACUACACUGCCU-3′). In certain embodiments such a passengerstrand has the following sequence from 5′ to 3′ in which one nucleotideis substituted with a different ribonucleotide (A, C, G, or U), asrepresented by N:

NCAAUCAGCUAACUACACUGCCU (SEQ ID NO: 268) GNAAUCAGCUAACUACACUGCCU(SEQ ID NO: 269) GCNAUCAGCUAACUACACUGCCU (SEQ ID NO: 270)GCANUCAGCUAACUACACUGCCU (SEQ ID NO: 271) GCAANCAGCUAACUACACUGCCU(SEQ ID NO: 272) GCAAUNAGCUAACUACACUGCCU (SEQ ID NO: 273)GCAAUCNGCUAACUACACUGCCU (SEQ ID NO: 274) GCAAUCANCUAACUACACUGCCU(SEQ ID NO: 275) GCAAUCAGNUAACUACACUGCCU (SEQ ID NO: 276)GCAAUCAGCNAACUACACUGCCU (SEQ ID NO: 277) GCAAUCAGCUNACUACACUGCCU(SEQ ID NO: 278) GCAAUCAGCUANCUACACUGCCU (SEQ ID NO: 279)GCAAUCAGCUAANUACACUGCCU (SEQ ID NO: 280) GCAAUCAGCUAACNACACUGCCU(SEQ ID NO: 281) GCAAUCAGCUAACUNCACUGCCU (SEQ ID NO: 282)GCAAUCAGCUAACUANACUGCCU (SEQ ID NO: 283) GCAAUCAGCUAACUACNCUGCCU(SEQ ID NO: 284) GCAAUCAGCUAACUACANUGCCU (SEQ ID NO: 285)GCAAUCAGCUAACUACACNGCCU (SEQ ID NO: 286) GCAAUCAGCUAACUACACUNCCU(SEQ ID NO: 287) GCAAUCAGCUAACUACACUGNCU (SEQ ID NO: 288)GCAAUCAGCUAACUACACUGCNU (SEQ ID NO: 289) GCAAUCAGCUAACUACACUGCCN(SEQ ID NO: 290)

In some embodiments, in addition to the single substitution shown above,there is a second substitution elsewhere in the sequence relative to SEQID NO:6. It is further contemplated that there may be a secondsubstitution with one of the substitutions described in a passengerstrand described above, or one or two deletions or insertions ofnucleotides in addition to the substitution described above.

In some embodiments, a passenger strand is 95-100% identical to SEQ IDNO:6, which should include the sequences disclosed above. Other examplesof such passenger strands include passenger strands with an insertion ofa single nucleotide, as discussed below, in which the followingsequences from 5′ to 3′ have an insertion of a nucleotide designated asN, which may be an A, C, G, or U:

NGCAAUCAGCUAACUACACUGCCU (SEQ ID NO: 291) GNCAAUCAGCUAACUACACUGCCU(SEQ ID NO: 292) GCNAAUCAGCUAACUACACUGCCU (SEQ ID NO: 293)GCANAUCAGCUAACUACACUGCCU (SEQ ID NO: 294) GCAANUCAGCUAACUACACUGCCU(SEQ ID NO: 295) GCAAUNCAGCUAACUACACUGCCU (SEQ ID NO: 296)GCAAUCNAGCUAACUACACUGCCU (SEQ ID NO: 297) GCAAUCANGCUAACUACACUGCCU(SEQ ID NO: 298) GCAAUCAGNCUAACUACACUGCCU (SEQ ID NO: 299)GCAAUCAGCNUAACUACACUGCCU (SEQ ID NO: 300) GCAAUCAGCUNAACUACACUGCCU(SEQ ID NO: 301) GCAAUCAGCUANACUACACUGCCU (SEQ ID NO: 302)GCAAUCAGCUAANCUACACUGCCU (SEQ ID NO: 303) GCAAUCAGCUAACNUACACUGCCU(SEQ ID NO: 304) GCAAUCAGCUAACUNACACUGCCU (SEQ ID NO: 305)GCAAUCAGCUAACUANCACUGCCU (SEQ ID NO: 306) GCAAUCAGCUAACUACNACUGCCU(SEQ ID NO: 307) GCAAUCAGCUAACUACANCUGCCU (SEQ ID NO: 308)GCAAUCAGCUAACUACACNUGCCU (SEQ ID NO: 309) GCAAUCAGCUAACUACACUNGCCU(SEQ ID NO: 310) GCAAUCAGCUAACUACACUGNCCU (SEQ ID NO: 311)GCAAUCAGCUAACUACACUGCNCU (SEQ ID NO: 312) GCAAUCAGCUAACUACACUGCCNU(SEQ ID NO: 313) GCAAUCAGCUAACUACACUGCCUN (SEQ ID NO: 314)

In some embodiments, in addition to the insertion in the passengerstrand shown above relative to SEQ ID NO:6, there may be a secondinsertion elsewhere in the strand. Combinations of insertions in thepassengers strands shown above are also contemplated for additionalpassenger strands.

Some embodiments concern an RNA molecule with an active strandcomprising SEQ ID NO:7. In specific embodiments, the active strandcomprises SEQ ID NO:9. Any of the embodiments discussed above in thecontext of active strands or strands with sequences that comprise,consist of or are a certain percentage identity to SEQ ID NOs:1, 2, or 5may be implemented in the context of a strand that comprises, consistsof or has a certain percentage identity to SEQ ID NOs: 7 or 9. SEQ IDNOs: 7 and 9 include each of SEQ ID NOs: 1, 2, and 5.

Certain embodiments concern an RNA molecule with a passenger strandcomprising SEQ ID NO:8. In specific embodiments, the passenger strandcomprises SEQ ID NO:10. Any of the embodiments discussed above in thecontext of passenger strands or strands with sequences that comprise,consist of, or are a certain percentage identity to SEQ ID NOs:3, 4, or6 may be implemented in the context of a strand that comprises, consistsof or has a certain percentage identity to SEQ ID NOs: 8 or 10. SEQ IDNOs: 8 and 10 include each of SEQ ID NOs: 3, 4, and 6.

The term “complementary region” or “complement” refers to a region of anucleic acid or mimic that is or is at least 60% complementary to themature, naturally occurring miRNA sequence. The complementary region isor is at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6,99.7, 99.8, 99.9 or 100% complementary, or any range derivable therein.With single polynucleotide sequences, there may be a hairpin loopstructure as a result of chemical bonding between the miRNA region andthe complementary region. In other embodiments, the complementary regionis on a different nucleic acid molecule than the miRNA region, in whichcase the complementary region is on the complementary strand and themiRNA region is on the active strand.

When the RNA molecule is a single polynucleotide, there can be a linkerregion between the miRNA region and the complementary region. In someembodiments, the single polynucleotide is capable of forming a hairpinloop structure as a result of bonding between the miRNA region and thecomplementary region. The linker constitutes the hairpin loop. It iscontemplated that in some embodiments, the linker region is, is atleast, or is at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, or 40 residues in length, or any range derivabletherein. In certain embodiments, the linker is between 3 and 30 residues(inclusive) in length.

A. Isolation of Nucleic Acids

Nucleic acids may be isolated using techniques well known to those ofskill in the art, though in particular embodiments, methods forisolating small nucleic acid molecules, and/or isolating RNA moleculescan be employed. Chromatography is a process often used to separate orisolate nucleic acids from protein or from other nucleic acids. Suchmethods can involve electrophoresis with a gel matrix, filter columns,capillary electrophoresis, alcohol precipitation, and/or otherchromatography. If miRNA from cells is to be used or evaluated, methodsgenerally involve lysing the cells with a chaotropic agent (e.g.,guanidinium isothiocyanate) and/or detergent (e.g., N-lauroyl sarcosine)prior to implementing processes for isolating particular populations ofRNA.

In particular methods for separating miRNA from other nucleic acids, agel matrix is prepared using polyacrylamide, though agarose can also beused. The gels may be graded by concentration or they may be uniform.Plates or tubing can be used to hold the gel matrix for electrophoresis.Usually one-dimensional electrophoresis is employed for the separationof nucleic acids. Plates are used to prepare a slab gel, while thetubing (glass or rubber, typically) can be used to prepare a tube gel.The phrase “tube electrophoresis” refers to the use of a tube or tubing,instead of plates, to form the gel. Materials for implementing tubeelectrophoresis can be readily prepared by a person of skill in the artor purchased, such as from C.B.S. Scientific Co., Inc. or Scie-Plas.

Methods may involve the use of organic solvents and/or alcohol toisolate nucleic acids, particularly miRNA used in methods andcompositions of the invention. Some embodiments are described in U.S.patent application Ser. No. 10/667,126, which is hereby incorporated byreference. Generally, this disclosure provides methods for efficientlyisolating small RNA molecules from cells comprising: adding an alcoholsolution to a cell lysate and applying the alcohol/lysate mixture to asolid support before eluting the RNA molecules from the solid support.In some embodiments, the amount of alcohol added to a cell lysateachieves an alcohol concentration of about 55% to 60%. While differentalcohols can be employed, ethanol works well. A solid support may be anystructure, and it includes beads, filters, and columns, which mayinclude a mineral or polymer support with electronegative groups. Aglass fiber filter or column has worked particularly well for suchisolation procedures.

In specific embodiments, miRNA isolation processes include: a) lysingcells in the sample with a lysing solution comprising guanidinium,wherein a lysate with a concentration of at least about 1 M guanidiniumis produced; b) extracting miRNA molecules from the lysate with anextraction solution comprising phenol; c) adding to the lysate analcohol solution for form a lysate/alcohol mixture, wherein theconcentration of alcohol in the mixture is between about 35% to about70%; d) applying the lysate/alcohol mixture to a solid support; e)eluting the miRNA molecules from the solid support with an ionicsolution; and, f) capturing the miRNA molecules. Typically the sample isdried down and resuspended in a liquid and volume appropriate forsubsequent manipulation.

B. Preparation of Nucleic Acids

Alternatively, nucleic acid synthesis is performed according to standardmethods. See, for example, Itakura and Riggs (1980). Additionally, U.S.Pat. Nos. 4,704,362, 5,221,619, and 5,583,013 each describe variousmethods of preparing synthetic nucleic acids. Non-limiting examples of asynthetic nucleic acid (e.g., a synthetic oligonucleotide), include anucleic acid made by in vitro chemical synthesis using phosphotriester,phosphite, or phosphoramidite chemistry and solid phase techniques suchas described in EP 266,032, incorporated herein by reference, or viadeoxynucleoside ri-phosphonate intermediates as described by Froehler etal., 1986 and U.S. Pat. No. 5,705,629, each incorporated herein byreference. In the methods described herein, one or more oligonucleotidemay be used. Oligonucleotide synthesis is well known to those of skillin the art. Various different mechanisms of oligonucleotide synthesishave been disclosed in for example, U.S. Pat. Nos. 4,659,774, 4,816,571,5,141,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146,5,602,244, each of which is incorporated herein by reference.

A non-limiting example of an enzymatically produced nucleic acidincludes one produced by enzymes in amplification reactions such as PCR™(see for example, U.S. Pat. Nos. 4,683,202 and 4,682,195, eachincorporated herein by reference), or the synthesis of anoligonucleotide described in U.S. Pat. No. 5,645,897, incorporatedherein by reference. Non-limiting examples of a biologically producednucleic acid include a recombinant nucleic acid produced (i.e.,replicated) in a living cell, such as a recombinant DNA vectorreplicated in bacteria or recombinant RNA or RNA vectors replicated inviruses (see for example, Sambrook et al., 2001, incorporated herein byreference).

Recombinant methods for producing nucleic acids in a cell are well knownto those of skill in the art. These include the use of vectors (viraland non-viral), plasmids, cosmids, and other vehicles for delivering anucleic acid to a cell, which may be the target cell (e.g., a cancercell) or simply a host cell (to produce large quantities of the desiredRNA molecule). Alternatively, such vehicles can be used in the contextof a cell free system so long as the reagents for generating the RNAmolecule are present. Such methods include those described in Sambrook,2003, Sambrook, 2001 and Sambrook, 1989, which are hereby incorporatedby reference.

In certain embodiments, the present invention concerns nucleic acidmolecules that are not synthetic. In some embodiments, the nucleic acidmolecule has a chemical structure of a naturally occurring nucleic acidand a sequence of a naturally occurring nucleic acid, such as the exactand entire sequence of a single stranded primary miRNA (see Lee, 2002),a single-stranded precursor miRNA, or a single-stranded mature miRNA.

II. THERAPEUTIC METHODS

Certain embodiments concern nucleic acids that perform the activities ofendogenous miR-34a or miR-34c when introduced into cells. In certainaspects, therapeutic nucleic acids (also referred to as nucleic acids)can be synthetic, non-synthetic, or a combination of synthetic andnon-synthetic miRNA sequences. Embodiments concern, in certain aspects,short nucleic acid molecules (therapeutic nucleic acids) that functionas miR-34a or miR-34c. The nucleic acid molecules are typicallysynthetic. The term “synthetic” refers to nucleic acid molecule that ischemically synthesized by a machine or apparatus and not producednaturally in a cell.

In certain aspects, RNA molecules may not have an entire sequence thatis identical or complementary to a sequence of a naturally-occurringmature miRNA. Such molecules may encompass all or part of anaturally-occurring sequence or a complement thereof. For example, asynthetic nucleic acid may have a sequence that differs from thesequence of a mature miRNA, but that altered sequence may provide one ormore functions that can be achieved with the natural sequence.

The term “isolated” means that the nucleic acid molecules are initiallyseparated from different molecules (in terms of sequence or structure)and unwanted nucleic acid molecules, such that a population of isolatednucleic acids is at least about 90% homogenous, and may be at leastabout 95, 96, 97, 98, 99, or 100% homogenous with respect to otherpolynucleotide molecules. In many aspects of the invention, a nucleicacid is isolated by virtue of it having been synthesized in vitroseparate from endogenous nucleic acids in a cell. It will be understood,however, that isolated nucleic acids may be subsequently mixed or pooledtogether.

In certain methods, there is a further step of administering theselected miRNA mimic or RNA molecule to a cell, tissue, organ, ororganism (collectively “biological matter”) in need of treatment relatedto modulation of the targeted miRNA or in need of the physiological orbiological results discussed herein (such as with respect to aparticular cellular pathway or result, for example, decrease in cellviability). Consequently, in some methods there is a step of identifyinga patient in need of treatment that can be provided by the miRNAmimic(s). It is contemplated that an effective amount of an miRNA mimiccan be administered in some embodiments. In particular embodiments,there is a therapeutic benefit conferred on the biological matter, wherea “therapeutic benefit” refers to an improvement in the one or moreconditions or symptoms associated with a disease or condition or animprovement in the prognosis, duration, or status with respect to thedisease. It is contemplated that a therapeutic benefit includes, but isnot limited to, a decrease in pain, a decrease in morbidity, a decreasein a symptom. For example, with respect to cancer, it is contemplatedthat a therapeutic benefit can be inhibition of tumor growth, preventionof metastasis, reduction in number of metastases, inhibition of cancercell proliferation, inhibition of cancer cell proliferation, inductionof cell death in cancer cells, inhibition of angiogenesis near cancercells, induction of apoptosis of cancer cells, reduction of cancer cellviability or number of viable cancer cells, reduction in pain, reductionin risk of recurrence, induction of chemo- or radiosensitivity in cancercells, prolongation of life, and/or delay of death directly orindirectly related to cancer.

In certain embodiments, an miRNA mimic is used to treat cancer. Cancerincludes, but is not limited to, malignant cancers, tumors, metastaticcancers, unresectable cancers, chemo- and/or radiation-resistantcancers, and terminal cancers.

Cancers that may be evaluated, diagnosed, and/or treated by methods andcompositions of the invention include cancer cells from the bladder,blood, bone, bone marrow, brain, breast, cardiovascular system, cervix,colon, connective tissue, endometrium, epithelium, esophagus, fat,gastrointestine, glands, gum, head, kidney, liver, lung, meninges,muscle, nasopharynx, neck, neurons, ovary, pancreas, prostate, rectum,retina, skin, spleen, stomach, testis, thymus, thyroid, tongue, oruterus. In addition, the cancer may specifically be of the followinghistological type, though it is not limited to these: neoplasm,malignant; carcinoma; carcinoma, undifferentiated; giant and spindlecell carcinoma; small cell carcinoma; papillary carcinoma; squamous cellcarcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrixcarcinoma; transitional cell carcinoma; papillary transitional cellcarcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma;hepatocellular carcinoma; combined hepatocellular carcinoma andcholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma;adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposiscoli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolaradenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clearcell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;papillary and follicular adenocarcinoma; nonencapsulating sclerosingcarcinoma; adrenal cortical carcinoma; endometroid carcinoma; skinappendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma;ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma;papillary cystadenocarcinoma; papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cellcarcinoma; infiltrating duct carcinoma; medullary carcinoma; lobularcarcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cellcarcinoma; adenosquamous carcinoma; adenocarcinoma w/squamousmetaplasia; thymoma, malignant; ovarian stromal tumor, malignant;thecoma, malignant; granulosa cell tumor, malignant; androblastoma,malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipidcell tumor, malignant; paraganglioma, malignant; extra-mammaryparaganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignantmelanoma; amelanotic melanoma; superficial spreading melanoma; maligmelanoma in giant pigmented nevus; epithelioid cell melanoma; bluenevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma,malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma;embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma;mixed tumor, malignant; mullerian mixed tumor; nephroblastoma;hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor,malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma,malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant;struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant;hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma;hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma;juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant;mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma;odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma,malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma;glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma;fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma;malignant lymphoma, small lymphocytic; malignant lymphoma, large cell,diffuse; malignant lymphoma, follicular; mycosis fungoides; otherspecified non-Hodgkin's lymphomas; malignant histiocytosis; multiplemyeloma; mast cell sarcoma; immunoproliferative small intestinaldisease; leukemia; lymphoid leukemia; plasma cell leukemia;erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia;basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mastcell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairycell leukemia. Moreover, miRNA mimics can be used for precancerouscells, such as those cells in metaplasia, dysplasia, and hyperplasia.

Methods include supplying or enhancing the activity of one or moremiRNAs in a cell. Methods also concern inducing certain cellularcharacteristics by providing to a cell a particular nucleic acid, suchas a specific therapeutic nucleic acid molecule, i.e., a miRNA mimicmolecule. The therapeutic miRNA mimic may have a sequence that isidentical to a naturally occurring miRNA with one or more designmodifications.

In certain aspects, a particular nucleic acid molecule provided to thecell is understood to correspond to a particular miRNA in the cell, andthus, the miRNA in the cell is referred to as the “corresponding miRNA.”In situations in which a named miRNA molecule is introduced into a cell,the corresponding miRNA will be understood to provide miRNA function. Itis contemplated in some embodiments, however, that the therapeuticnucleic acid introduced into a cell is not a mature miRNA but is capableof becoming or functioning as a mature miRNA under the appropriatephysiological conditions. In cases in which a particular correspondinggene or gene transcript is being targeted by an miRNA mimic, theparticular gene or gene transcript will be referred to as the “targetedgene.” It is contemplated that multiple corresponding genes may betargeted by one or more different miRNA mimics. In particularembodiments, more than one therapeutic nucleic acid is introduced into acell. Moreover, in other embodiments, more than one miRNA mimic isintroduced into a cell. Furthermore, a combination of therapeuticnucleic acid(s) may be introduced into a cell. The inventors contemplatethat a combination of therapeutic nucleic acids may act at one or morepoints in cellular pathways of cells and that such combination may haveincreased efficacy on the target cell while not adversely affectingnormal or non-targeted cells. Thus, a combination of therapeutic nucleicacids may have a minimal adverse effect on a subject or patient whilesupplying a sufficient therapeutic effect, such as amelioration of acondition, growth inhibition of a cell, death of a targeted cell,alteration of cell phenotype or physiology, slowing of cellular growth,sensitization to a second therapy, sensitization to a particulartherapy, and the like.

Methods include identifying a cell or patient in need of inducing thosetherapeutics effects or cellular characteristics. Also, it will beunderstood that an amount of a therapeutic nucleic acid that is providedto a cell or organism is an “effective amount,” which refers to anamount needed (or a sufficient amount) to achieve a desired goal, suchas inducing a particular therapeutic effect or cellularcharacteristic(s) or reducing cancer growth or killing cancer cells oralleviating symptoms associated with a cancer.

In certain aspects methods can include providing or introducing into acell a nucleic acid molecule corresponding to a mature miRNA in the cellin an amount effective to achieve a desired physiological result.Moreover, methods can involve providing multiple synthetic therapeuticnucleic acids. It is contemplated that in these embodiments, methods mayor may not be limited to providing only one or more synthetic molecules.In this situation, a cell or cells may be provided with a syntheticmolecule corresponding to a particular miRNA and a synthetic moleculecorresponding to a different miRNA. Furthermore, any method articulatedusing a list of miRNA targets using Markush group language may bearticulated without the Markush group language and a disjunctive article(i.e., or) instead, and vice versa.

In some embodiments, there is a method for reducing or inhibiting cellproliferation, propagation, or renewal in a cell comprising introducinginto or providing to the cell an effective amount of (i) a therapeuticnucleic acid or (ii) a synthetic molecule that corresponds to a miRNAsequence. In certain embodiments the methods involve introducing intothe cell an effective amount of (i) a miRNA mimic molecule having a 5′to 3′ sequence that is at least 90% identical to the 5′ to 3′ sequenceof one or more mature miRNA.

Certain aspects of the invention include methods of treating apathologic condition, such as cancer or precancerous conditions. In oneaspect, the method comprises contacting a target cell with one or morenucleic acids comprising at least one nucleic acid segment having all ora portion of a miRNA sequence or a complement thereof. The segment maybe 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30 or more nucleotides or nucleotide analog, including allintegers there between. Some embodiments includes the modulation of geneexpression, miRNA expression or function or mRNA expression or functionwithin a target cell, such as a cancer cell.

Typically, an endogenous gene, miRNA or mRNA is modulated in the cell.In certain aspects, a therapeutic nucleic acid sequence comprises atleast one segment that is at least 70, 75, 80, 85, 90, 95, or 100%identical in nucleic acid sequence to one or more miRNA or gene sequenceor complement thereof. Modulation of the expression or processing of agene, miRNA, or mRNA of the cell or a virus can be through modulation ofthe processing of a nucleic acid, such processing includingtranscription, transportation and/or translation within a cell.Modulation may also be effected by the inhibition or enhancement ofmiRNA activity with a cell, tissue, or organ. Such processing may affectthe expression of an encoded product or the stability of the mRNA.

It will be understood in methods of the invention that a cell or otherbiological matter such as an organism (including patients) can beprovided a therapeutic nucleic acid corresponding to or targeting aparticular miRNA by administering to the cell or organism a nucleic acidmolecule that functions as the corresponding miRNA once inside the cell.Thus, it is contemplated that a nucleic acid is provided such that itbecomes processed into a mature and active miRNA once it has access tothe cell's processing machinery. In certain aspects, it is specificallycontemplated that the miRNA molecule provided is not a mature moleculebut a nucleic acid molecule that can be processed into the mature miRNAor its functional equivalent once it is accessible to processingmachinery.

The teem “non-synthetic” in the context of miRNA means that the miRNA isnot “synthetic,” as defined herein. Furthermore, it is contemplated thatin embodiments of the invention that concern the use of syntheticmiRNAs, the use of corresponding non-synthetic miRNAs is also consideredan aspect of the invention, and vice versa. It will be understood thatthe term “providing” an agent is used to include “administering” theagent to a patient.

In certain embodiments, methods also include targeting an miRNA in acell or organism. The term “corresponding to a miRNA” means a nucleicacid will be employed so as to mimic (provide the activity or functionof) a selected miRNA. In some embodiments the regulation is achievedwith a synthetic or non-synthetic nucleic acid that corresponds to thetargeted miRNA, which effectively provides the function of the targetedmiRNA to the cell or organism (positive regulation).

Furthermore, it is contemplated that the nucleic acid compositions maybe provided as part of a therapy to a patient, in conjunction withtraditional therapies or preventative agents. Moreover, it iscontemplated that any method discussed in the context of therapy may beapplied preventatively, particularly in a patient identified to bepotentially in need of the therapy or at risk of the condition ordisease for which a therapy is needed.

In addition, methods of the invention concern employing one or morenucleic acid corresponding to a miRNA and a therapeutic drug. Thenucleic acid can enhance the effect or efficacy of the drug, reduce anyside effects or toxicity, modify its bioavailability, and/or decreasethe dosage or frequency needed. In certain embodiments, the therapeuticdrug is a cancer therapeutic. Consequently, in some embodiments, thereis a method of treating a precancer or cancer in a patient comprisingadministering to the patient a cancer therapeutic (i.e., a secondtherapeutic) and an effective amount of at least one nucleic acidmolecule that improves the efficacy of the cancer therapeutic orprotects non-cancer cells. Cancer therapies also include a variety ofcombination therapies with both chemical and radiation based treatments.

Generally, miRNA mimics can be given to decrease the activity of anucleic acid targeted by the miRNA. Methods generally contemplatedinclude providing or introducing one or more different nucleic acidmolecules corresponding to one or more different genes. It iscontemplated that the following, at least the following, or at most thefollowing number of different nucleic acid or miRNA molecules may bedetected, assessed, provided or introduced: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, or more, including any value or range derivable there between.

III. PHARMACEUTICAL FORMULATIONS AND DELIVERY

Methods include the delivery of an effective amount of a therapeuticnucleic acid comprising or consisting essentially of a mature miRNAsequence. An “effective amount” of the pharmaceutical composition,generally, is defined as that amount sufficient to detectably andrepeatedly to achieve the stated desired result, for example, toameliorate, reduce, minimize or limit the extent of the disease or itssymptoms. Other more rigorous definitions may apply, includingelimination, eradication or cure of disease.

In certain embodiments, it is desired to kill cells, inhibit cellgrowth, inhibit metastasis, decrease tumor or tissue size, and/orreverse or reduce the malignant or disease phenotype of cells. Theroutes of administration will vary, naturally, with the location andnature of the lesion or site to be targeted, and include, e.g.,intradermal, subcutaneous, regional, parenteral, intravenous,intramuscular, intranasal, systemic, and oral administration andformulation. Injection or perfusion of a therapeutic nucleic acid isspecifically contemplated for discrete, solid, accessible precancers orcancers, or other accessible target areas. Local, regional, or systemicadministration also may be appropriate.

In the case of surgical intervention, the present invention may be usedpreoperatively, to render an inoperable lesion subject to resection.Alternatively, the present invention may be used at the time of surgery,and/or thereafter, to treat residual or metastatic disease. For example,a resected tumor bed may be injected or perfused with a formulationcomprising a therapeutic nucleic acid or combinations thereof.Administration may be continued post-resection, for example, by leavinga catheter implanted at the site of the surgery. Periodic post-surgicaltreatment also is envisioned. Continuous perfusion of a nucleic acidalso is contemplated.

Continuous administration also may be applied where appropriate, forexample, where a tumor or other undesired affected area is excised andthe tumor bed or targeted site is treated to eliminate residual,microscopic disease. Delivery via syringe or catherization iscontemplated. Such continuous perfusion may take place for a period fromabout 1-2 hours, to about 2-6 hours, to about 6-12 hours, to about 12-24hours, to about 1-2 days, to about 1-2 wk or longer following theinitiation of treatment. Generally, the dose of the therapeuticcomposition via continuous perfusion will be equivalent to that given bya single or multiple injections, adjusted over a period of time duringwhich the perfusion occurs.

Treatment regimens may vary as well and often depend on the type and/orlocation of a lesion, the target site, disease progression, and thehealth, immune condition, and age of the patient. Certain tumor typeswill require more aggressive treatment. The clinician will be bestsuited to make such decisions based on the known efficacy and toxicity(if any) of the therapeutic formulations.

In certain embodiments, the lesion or affected area being treated maynot, at least initially, be resectable. Treatments with compositions ofthe invention may increase the respectability of the lesion due toshrinkage at the margins or by elimination of certain particularlyinvasive portions. Following treatments, resection may be possible.Additional treatments subsequent to resection may serve to eliminatemicroscopic residual disease at the tumor or targeted site.

Treatments may include various “unit doses.” A unit dose is defined ascontaining a predetermined quantity of a therapeutic composition(s). Thequantity to be administered, and the particular route and formulation,are within the skill of those in the clinical arts. A unit dose need notbe administered as a single injection but may comprise continuousinfusion over a set period of time. A unit dose may conveniently bedescribed in terms of ng, μg, or mg of miRNA or miRNA mimetic.Alternatively, the amount specified may be the amount administered asthe average daily, average weekly, or average monthly dose.

A therapeutic nucleic acid can be administered to the patient in a doseor doses of about or of at least about 0.5, 1, 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450,460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590,600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730,740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870,880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 ng, μgor mg, or more, or any range derivable therein. Alternatively, theamount specified may be the amount administered as the average daily,average weekly, or average monthly dose, or it may be expressed in termsof mg/kg, where kg refers to the weight of the patient and the mg isspecified above. In other embodiments, the amount specified is anynumber discussed above but expressed as mg/m² (with respect to tumorsize or patient surface area). In some embodiments, a dose or regimenmay be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, and/or 1, 2, 3, 4, 5, 6, 7day(s), and/or 1, 2, 3, 4 weeks, and any range derivable therein, to apatient in need of treatment.

In some embodiments, the method for the delivery of a therapeuticnucleic acid is via local or systemic administration. However, thepharmaceutical compositions disclosed herein may also be administeredparenterally, subcutaneously, intratracheally, intravenously,intradermally, intramuscularly, or even intraperitoneally as describedin U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 (each specificallyincorporated herein by reference in its entirety).

Injection of nucleic acids may be delivered by syringe or any othermethod used for injection of a solution, as long as the nucleic acid andany associated components can pass through the particular gauge ofneedle required for injection. A syringe system has also been describedfor use in gene therapy that permits multiple injections ofpredetermined quantities of a solution precisely at any depth (U.S. Pat.No. 5,846,225).

Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, mixtures thereof, andin oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). Typically, the form must be sterile andmust be fluid to the extent that easy syringability exists. It must bestable under the conditions of manufacture and storage and preservedagainst the contaminating action of microorganisms, such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (e.g., glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and/or vegetable oils. Proper fluidity may be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

In certain formulations, a water-based formulation is employed while inothers, it may be lipid-based. In particular embodiments of theinvention, a composition comprising a nucleic acid of the invention isin a water-based formulation. In other embodiments, the formulation islipid based.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, intratumoral, intralesional, andintraperitoneal administration. In this connection, sterile aqueousmedia which can be employed will be known to those of skill in the artin light of the present disclosure. For example, one dosage may bedissolved in 1 ml of isotonic NaCl solution and either added to 1000 mlof hypodermoclysis fluid or injected at the proposed site of infusion,(see for example, “Remington's Pharmaceutical Sciences” 15th Edition,pages 1035-1038 and 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

As used herein, a “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human.

The nucleic acid(s) are administered in a manner compatible with thedosage formulation, and in such amount as will be therapeuticallyeffective. The quantity to be administered depends on the subject to betreated, including, e.g., the aggressiveness of the disease or cancer,the size of any tumor(s) or lesions, the previous or other courses oftreatment. Precise amounts of active ingredient required to beadministered depend on the judgment of the practitioner. Suitableregimes for initial administration and subsequent administration arealso variable, but are typified by an initial administration followed byother administrations. Such administration may be systemic, as a singledose, continuous over a period of time spanning 10, 20, 30, 40, 50, 60minutes, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24 or more hours, and/or 1, 2, 3, 4, 5, 6,7, days or more. Moreover, administration may be through a time releaseor sustained release mechanism, implemented by formulation and/or modeof administration.

Other delivery systems suitable include, but are not limited to,time-release, delayed release, sustained release, or controlled releasedelivery systems. Such systems may avoid repeated administrations inmany cases, increasing convenience to the subject and the physician.Many types of release delivery systems are available and known to thoseof ordinary skill in the art. They include, for example, polymer-basedsystems such as polylactic and/or polyglycolic acids, polyanhydrides,polycaprolactones, copolyoxalates, polyesteramides, polyorthoesters,polyhydroxybutyric acid, and/or combinations of these. Microcapsules ofthe foregoing polymers containing nucleic acids are described in, forexample, U.S. Pat. No. 5,075,109. Other examples include nonpolymersystems that are lipid-based including sterols such as cholesterol,cholesterol esters, and fatty acids or neutral fats such as mono-, di-and triglycerides; hydrogel release systems; liposome-based systems;phospholipid based-systems; silastic systems; peptide based systems; waxcoatings; compressed tablets using conventional binders and excipients;or partially fused implants. Specific examples include, but are notlimited to, erosional systems in which the RNA molecule is contained ina formulation within a matrix (for example, as described in U.S. Pat.Nos. 4,452,775, 4,675,189, 5,736,152, 4,667,013, 4,748,034 and5,239,660, which are hereby incorporated by reference), or diffusionalsystems in which an active component controls the release rate (forexample, as described in U.S. Pat. Nos. 3,832,253, 3,854,480, 5,133,974and 5,407,686, which are hereby incorporated by reference). Theformulation may be as, for example, microspheres, hydrogels, polymericreservoirs, cholesterol matrices, or polymeric systems. In someembodiments, the system may allow sustained or controlled release of thecomposition to occur, for example, through control of the diffusion orerosion/degradation rate of the formulation containing the RNAmolecules. In addition, a pump-based hardware delivery system may beused to deliver one or more embodiments.

Examples of systems in which release occurs in bursts includes, e.g.,systems in which the composition is entrapped in liposomes which areencapsulated in a polymer matrix, the liposomes being sensitive tospecific stimuli, e.g., temperature, pH, light or a degrading enzyme andsystems in which the composition is encapsulated by an ionically-coatedmicrocapsule with a microcapsule core degrading enzyme. Examples ofsystems in which release of the inhibitor is gradual and continuousinclude, e.g., erosional systems in which the composition is containedin a form within a matrix and effusional systems in which thecomposition permeates at a controlled rate, e.g., through a polymer.Such sustained release systems can be e.g., in the form of pellets, orcapsules.

Compositions and methods can be used to enhance delivery of RNAmolecules (see Shim and Kwon (2010), which is incorporated herein byreference, for review). Compositions and methods for enhanced deliverycan provide for efficient delivery through the circulation, appropriatebiodistribution, efficient cellular transport, efficient intracellularprocessing, and the like. Formulations and compositions can include, butare not limited to one or more of chemical modification of RNAmolecules, incorporation of an RNA molecule or RNA precursors into aviral or non-viral vector, targeting of RNA molecule delivery, and/orcoupling RNA molecules with a cellular delivery enhancer.

In certain aspects chemically modified RNA molecules can include, withor without the chemical modifications discussed above, the conjugationof an RNA molecule to a carrier molecule. In certain aspects the carriermolecule is a natural or synthetic polymer. For example, a carriermolecule can be cholesterol or an RNA aptamer and the like. A carriermolecule can be conjugated to the RNA molecules at the 5′ and/or 3′ endof either the active or passenger strand, or at an internal nucleotideposition. The carrier can be conjugated the either strand of an RNAmolecules.

In a further aspect one or two strands of the RNA molecule can beencoded by or delivered with a viral vector. A variety of viral vectorsknow in the art can be modified to express or carry an RNA molecule in atarget cell, for example herpes simplex virus-1 or lentiviral vectorshave been used to enhance the delivery of siRNA.

In a still further aspect, an RNA molecule can be associated with anon-viral vector. Non-viral vectors can be coupled to targeting anddelivery enhancing moieties, such as antibodies, various polymers (e.g.,PEG), fusogenic peptides, linkers, cell penetrating peptides and thelike. Non-viral vectors include, but are not limited to liposomes andlipoplexes, polymers and peptides, synthetic particles and the like. Incertain aspects a liposome or lipoplex has a neutral, negative orpositive charge and can comprise cardolipin, anisamide-conjugatedpolyethylene glycol, dioleoyl phosphatidylcholine, or a variety of otherneutral, anionic, or cationic lipids or lipid conjugates. siRNAs can becomplexed to cationic polymers (e.g., polyethylenimine (PEI)),biodegradable cationic polysaccharide (e.g., chitosan), or cationicpolypeptides (e.g., atelocollagen, poly lysine, and protamine).

In certain aspects RNA delivery can be enhanced by targeting the RNA toa cell. Targeting moieties can be conjugated to a variety of deliverycompositions and provide selective or specific binding to a targetcell(s). Targeting moieties can include, but are not limited to moietiesthat bind to cell surface receptors, cell specific extracellularpolypeptide, saccharides or lipids, and the like. For example, smallmolecules such as folate, peptides such as RGD containing peptides, andantibodies such as antibodies to epidermal growth factor receptor can beused to target specific cell types.

In a further aspect, delivery can be enhanced by moieties that interactwith cellular mechanisms and machinery, such as uptake and intracellulartrafficking. In certain aspects cell penetrating peptides (CPPs) (e.g.,TAT and MPG from HIV-1, penetratin, polyarginine can be coupled with ansiRNA or a delivery vector to enhance delivery into a cell. Fusogenicpeptides (e.g., endodomain derivatives of HIV-1 envelope (HGP) orinfluenza fusogenic peptide (diINF-7)) can also be used to enhancecellular delivery.

A variety of delivery systems such as cholesterol-siRNA, RNAaptamers-siRNA, adenoviral vector, lentiviral vector, stable nucleicacid lipid particle (SNALP), cardiolipin analog-based liposome,DSPE-polyethylene glycol-DOTAP-cholesterol liposome, hyaluronan-DPPEliposome, neutral DOPC liposome, atelocollagen, chitosan,polyethylenimine, poly-lysine, protamine, RGD-polyethyleneglycol-polyethylenimine, HER-2 liposome with histidine-lysine peptide,HIV antibody-protamine, arginine, oligoarginine (9R) conjugated watersoluble lipopolymer (WSLP), oligoarginine (15R), TAT-PAMAM,cholesterol-MPG-8, DOPE-cationic liposome, GALA peptide-PEG-MMP-2cleavable peptide-DOPE and the like have been used to enhance thedelivery of siRNA.

The optimal therapeutic dose range for the miRNA mimics in cancerpatients is contemplated to be 0.01-5.0 mg of miRNA per kg of patientbody weight (mg/kg). In some embodiments, it is contemplated that about,at least about, or at most about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06,0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7. 3.8, 3.9,4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 mg of an RNAmolecule, or any range derivable therein, may be formulated in acomposition and/or administered to a patient. In some embodiments, apatient may be administered 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07,0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7. 3.8, 3.9, 4.0,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 mg/kg of an RNAmolecule, or any range derivable therein, per dose or regimen, which maybe administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24 hours, and/or 1, 2, 3, 4, 5, 6, 7day(s), and/or 1, 2, 3, 4 weeks, and any range derivable therein.

Injections can be intravenous (IV), intraperitoneal (IP), intramuscular(IM), intratumoral (IT), intratracheally (for pulmonary delivery),intravitreal (for eye diseases), or subcutaneous, all of which have beendetermined to be effective as a delivery method for RNA moleculesdescribed herein. Several delivery technologies are specificallycontemplated, including, but not limited to, neutral lipid emulsion,(NLE), atelocollagen, SNALP, DiLA, and cyclodextrin, which are discussedin further detail below.

Neutral lipid emulsions (NLEs) are a collection of formulations thatcombine a neutral lipid, oil, and emulsifier with a miRNA mimic toproduce complexes that enable delivery of miRNAs to tumors and othertissues following intravenous (IV) injection. One such formulationcombines 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), squalene,Polysorbate 20 (Tween-20), and ascorbic acid at a ratio of1:2.6:53.4:0.1 (w/w). The NLE components are mixed in a solvent likechloroform and then the solvent is removed using a rotary evaporatorleaving a viscous solution. A miRNA mimic dissolved in PBS is added at aratio of 1:2 (w/w) to the DOPC. In certain embodiments, the ratio of RNAmolecule to DOPC is about, at least about, or at most about 0.1:1,0:2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1:0.9,1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4; 1:0.3, 1:0.2, 1:0.1 and any rangederivable therein. Sonication produces particles+miRNA that can be IVinjected at rates of approximately 0.01-1 mg/kg in humans. In certainembodiments, the amount of this formulation is provided to a patient inamounts of 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09,0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7. 3.8, 3.9, 4.0, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 mg/kg to a patient per dose orregimen, which may be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, and/or 1,2, 3, 4, 5, 6, 7 day(s), and/or 1, 2, 3, 4 weeks, and any rangederivable therein.

Atelocollagen/miRNA complexes are prepared by mixing equal volumes ofatelocollagen (0.1% in PBS at pH 7.4) (Koken Co., Ltd.; Tokyo, Japan)and miRNA solution (20 μM miRNA) and rotating the mixtures for 1 hr at4° C. The resulting miRNA/atelocollagen complexes are diluted in PBS toa final concentration of atelocollagen of 0.05%. In certain embodiments,the final percentage concentration of atelocollagen is about, about atleast, or about at most 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%,0.08%, 0.09%, 0.10%, 0.11%, 0.10%, 0.11%, 0.12%, 0.13, 0.14%, 0.15%,0.16%, 0.17%, 0.18%, 0.19%, 0.20%, and any range derivable therein.

SNALP categorizes a collection of formulations developed by TekmiraPharmaceutical Corp. (Burnaby, BC, CA) for the systemic delivery ofnucleic acids. The most published formulation contains the lipids3-N-[(qmethoxypoly(ethyleneglycol)2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA),1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a2:40:10:48 molar percent ratio. The lipid formulation is mixed withsiRNA/miRNA and forms particles using the ethanol dilution method (Jeffs2005, which is hereby incorporated by reference). In some embodiments,the ratio of lipid to nucleic acid (w:w) is, is at least, or is at mostabout 0.001:1, 0.002:1, 0.003:1, 0.004:1, 0.005:1, 0.006:1, 0.007:1,0.008:1, 0.009:1, 0.01:1, 0.02:1, 0.03:1, 0.04:1, 0.05:1, 0.06:1,0.07:1, 0.08:1, 0.09:1, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1,0.8:1, 0.9:1, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4; 1:0.3,1:0.2, 1:0.1, 1:0.09, 1:0.08, 1:0.07, 1:0.06, 1:0.05, 1:0.04, 1:0.03,1:0.02, 1:0.01, 1:0.009, 1:0.008, 1:0.007, 1:0.006, 1:0.005, 1:0.004,1:0.003, 1:0.002, 1:0.001, or any range derivable therein.

Tekmira claims to achieve greater than 90% encapsulation efficiency.Particle sizes are approximately 110 nm.

DiLA² describes a group of a variety of formulations developed by MarinaBiotech Inc. (Bothell, Wash., USA) for the systemic delivery of small,dsRNAs. One formulation combines C18:1-norArg-NH₃Cl—C16, cholesterylhemisuccinate (CHEMS, Anatrace, CH210), cholesterol (Anatrace CH200),and DMPE-PEG2k (Genzyme) at ratios of 50:28:20:2 (w/w). A small, dsRNAis combined with the lipid formulation at an RNA:lipid ratio of between1.7:1 to 5:1 (w/w). In some embodiments, the RNA:lipid ratio is about,at least about, or at most about 0.001:1, 0.002:1, 0.003:1, 0.004:1,0.005:1, 0.006:1, 0.007:1, 0.008:1, 0.009:1, 0.01:1, 0.02:1, 0.03:1,0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.1:1, 0.2:1, 0.3:1,0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1:0.9, 1:0.8, 1:0.7,1:0.6, 1:0.5, 1:0.4; 1:0.3, 1:0.2, 1:0.1, 1:0.09, 1:0.08, 1:0.07,1:0.06, 1:0.05, 1:0.04, 1:0.03, 1:0.02, 1:0.01, 1:0.009, 1:0.008,1:0.007, 1:0.006, 1:0.005, 1:0.004, 1:0.003, 1:0.002, 1:0.001, and anyrange derivable therein. The two components are mixed via an impingingstream and incubated for 1 hour to produce stable particles withdiameters of approximately 125 nm.

Calando Pharmaceuticals, Inc. (Pasadena, Calif., USA) has developed adelivery platform called RONDEL™ that features cyclodextrin-basedparticles. Cyclodextrin polycations (CDP) are mixed with anadamantane-PEG5000 (AD-PEG) conjugate at a 1:1 AD:CDP (mol/mol) ratio(Hu-Lieskovan 2005, which is hereby incorporated by reference).Transferrin-modified AD-PEG (AD-PEG-transferrin) can be added at a1:1,000 AD-PEG-transferrin:AD-PEG (w/w) ratio to provide a targetingmoiety to improve delivery to cancer cells with elevated transferrinlevels. The mixture is added to an equal volume of RNA molecule at acharge ratio (positive charges from CDP to negative charges from miRNAbackbone) of 3:1 (+/−). In certain embodiments, the charge ratio betweenthe mixture and RNA molecules is about, at least about, or at most about5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, or any range derivabletherein, An equal volume of 10% (w/v) glucose in water is added to theresulting polyplexes to give a final polyplex formulation in 5% (w/v)glucose (D5W) suitable for injection.

In some embodiments, particles are used to delivery a therapeuticnucleic acid. In some embodiments, the particle size is about, at leastabout, or at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 nm,or any range derivable therein.

A. Combination Treatments

In certain embodiments, the compositions and methods involve atherapeutic nucleic acid. These compositions can be used in combinationwith a second therapy to enhance the effect of the miRNA therapy, orincrease the therapeutic effect of another therapy being employed. Thesecompositions would be provided in a combined amount effective to achievethe desired effect, such as the killing of a cancer cell and/or theinhibition of cellular hyperproliferation. This process may involvecontacting the cells with the therapeutic nucleic acid or second therapyat the same or different time. This may be achieved by contacting thecell with one or more compositions or pharmacological formulation thatincludes or more of the agents, or by contacting the cell with two ormore distinct compositions or formulations, wherein one compositionprovides (1) therapeutic nucleic acid; and/or (2) a second therapy. Asecond composition or method may be administered that includes achemotherapy, radiotherapy, surgical therapy, immunotherapy or genetherapy.

It is contemplated that one may provide a patient with the miRNA therapyand the second therapy within about 12-24 h of each other and, morepreferably, within about 6-12 h of each other. In some situations, itmay be desirable to extend the time period for treatment significantly,however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

In certain embodiments, a course of treatment will last 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 days or more. It iscontemplated that one agent may be given on day 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, and/or 90, any combination thereof,and another agent is given on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, and/or 90, or any combination thereof. Within asingle day (24-hour period), the patient may be given one or multipleadministrations of the agent(s). Moreover, after a course of treatment,it is contemplated that there is a period of time at which no treatmentis administered. This time period may last 1, 2, 3, 4, 5, 6, 7 days,and/or 1, 2, 3, 4, 5 weeks, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12months or more, depending on the condition of the patient, such as theirprognosis, strength, health, etc.

Administration of any compound or therapy of the present invention to apatient will follow general protocols for the administration of suchcompounds, taking into account the toxicity, if any, of the vector orany protein or other agent. Therefore, in some embodiments there is astep of monitoring toxicity that is attributable to combination therapy.It is expected that the treatment cycles would be repeated as necessary.It also is contemplated that various standard therapies, as well assurgical intervention, may be applied in combination with the describedtherapy.

In specific aspects, it is contemplated that a second therapy, such aschemotherapy, radiotherapy, immunotherapy, surgical therapy or othergene therapy, is employed in combination with the miRNA therapy, asdescribed herein.

Examples of chemotherapeutic agents include alkylating agents such asthiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB 1-TM 1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammail and calicheamicin omega1; dynemicin, including dynemicin A; bisphosphonates, such asclodronate; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antiobiotic chromophores, aclacinomysins,actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin,carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharidecomplex); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonicacid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes(especially T-2 toxin, verracurin A, roridin A and anguidine); urethan;vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide;thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumcoordination complexes such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11);topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO);retinoids such as retinoic acid; capecitabine; and pharmaceuticallyacceptable salts, acids or derivatives of any of the above.

Radiotherapy, also called radiation therapy, is the treatment of cancerand other diseases with ionizing radiation. Ionizing radiation depositsenergy that injures or destroys cells in the area being treated bydamaging their genetic material, making it impossible for these cells tocontinue to grow. Although radiation damages both cancer cells andnormal cells, the latter are able to repair themselves and functionproperly. Radiotherapy may be used to treat localized solid tumors, suchas cancers of the skin, tongue, larynx, brain, breast, or cervix. It canalso be used to treat leukemia and lymphoma (cancers of theblood-forming cells and lymphatic system, respectively).

Radiation therapy used according to the present invention may include,but is not limited to, the use of γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves, proton beamirradiation (U.S. Pat. Nos. 5,760,395 and 4,870,287) and UV-irradiation.It is most likely that all of these factors effect a broad range ofdamage on DNA, on the precursors of DNA, on the replication and repairof DNA, and on the assembly and maintenance of chromosomes. Dosageranges for X-rays range from daily doses of 50 to 200 roentgens forprolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000roentgens. Dosage ranges for radioisotopes vary widely, and depend onthe half-life of the isotope, the strength and type of radiationemitted, and the uptake by the neoplastic cells. Radiotherapy maycomprise the use of radiolabeled antibodies to deliver doses ofradiation directly to the cancer site (radioimmunotherapy). Onceinjected into the body, the antibodies actively seek out the cancercells, which are destroyed by the cell-killing (cytotoxic) action of theradiation. This approach can minimize the risk of radiation damage tohealthy cells.

Stereotactic radio-surgery (gamma knife) for brain and other tumors doesnot use a knife, but very precisely targeted beams of gamma radiotherapyfrom hundreds of different angles. Only one session of radiotherapy,taking about four to five hours, is needed. For this treatment aspecially made metal frame is attached to the head. Then, several scansand x-rays are carried out to find the precise area where the treatmentis needed. During the radiotherapy for brain tumors, the patient lieswith their head in a large helmet, which has hundreds of holes in it toallow the radiotherapy beams through. Related approaches permitpositioning for the treatment of tumors in other areas of the body.

In the context of cancer treatment, immunotherapeutics, generally, relyon the use of immune effector cells and molecules to target and destroycancer cells. Trastuzumab (Herceptin™) is such an example. The immuneeffector may be, for example, an antibody specific for some marker onthe surface of a tumor cell. The antibody alone may serve as an effectorof therapy or it may recruit other cells to actually affect cellkilling. The antibody also may be conjugated to a drug or toxin(chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussistoxin, etc.) and serve merely as a targeting agent. Alternatively, theeffector may be a lymphocyte carrying a surface molecule that interacts,either directly or indirectly, with a tumor cell target. Variouseffector cells include cytotoxic T cells and NK cells. The combinationof therapeutic modalities, i.e., direct cytotoxic activity andinhibition or reduction of ErbB2 would provide therapeutic benefit inthe treatment of ErbB2 overexpressing cancers.

In one aspect of immunotherapy, the tumor or disease cell must bear somemarker that is amenable to targeting, i.e., is not present on themajority of other cells. Many tumor markers exist and any of these maybe suitable for targeting in the context of the present invention.Common tumor markers include carcinoembryonic antigen, prostate specificantigen, urinary tumor associated antigen, fetal antigen, tyrosinase(p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP,estrogen receptor, laminin receptor, erb B and p155. An alternativeaspect of immunotherapy is to combine anticancer effects with immunestimulatory effects. Immune stimulating molecules also exist including:cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines suchas MIP-1, MCP-1, IL-8 and growth factors such as FLT3 ligand. Combiningimmune stimulating molecules, either as proteins or by using genedelivery in combination with a tumor suppressor such as MDA-7 has beenshown to enhance anti-tumor effects (Ju et al., 2000). Moreover,antibodies against any of these compounds can be used to target theanti-cancer agents discussed herein.

Examples of immunotherapies currently under investigation or in use areimmune adjuvants e.g., Mycobacterium bovis, Plasmodium falciparum,dinitrochlorobenzene and aromatic compounds (U.S. Pat. Nos. 5,801,005and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998),cytokine therapy e.g., interferons α, β and γ; IL-1, GM-CSF and TNF(Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998)gene therapy e.g., TNF, IL-1, IL-2, p53 (Qin et al., 1998; Austin-Wardand Villaseca, 1998; U.S. Pat. Nos. 5,830,880 and 5,846,945) andmonoclonal antibodies e.g., anti-ganglioside GM2, anti-HER-2, anti-p185;Pietras et al., 1998; Hanibuchi et al., 1998; U.S. Pat. No. 5,824,311).Herceptin (trastuzumab) is a chimeric (mouse-human) monoclonal antibodythat blocks the HER2-neu receptor. It possesses anti-tumor activity andhas been approved for use in the treatment of malignant tumors (Dillman,1999). A non-limiting list of several known anti-cancerimmunotherapeutic agents and their targets includes (GenericName/Target) Cetuximab/EGFR, Panitumuma/EGFR, Trastuzumab/erbB2receptor, Bevacizumab/VEGF, Alemtuzumab/CD52, Gemtuzumabozogamicin/CD33, Rituximab/CD20, Tositumomab/CD20, Matuzumab/EGFR,Ibritumomab tiuxetan/CD20, Tositumomab/CD20, HuPAM4/MUC 1,MORAb-009/Mesothelin, G250/carbonic anhydrase IX, mAb 8H9/8H9 antigen,M195/CD33, Ipilimumab/CTLA4, HuLuc63/CS1, Alemtuzumab/CD53,Epratuzumab/CD22, BC8/CD45, HuJ591/Prostate specific membrane antigen,hA20/CD20, Lexatumumab/TRAIL receptor-2, Pertuzumab/HER-2 receptor,Mik-beta-1/IL-2R, RAV12/RAAG12, SGN-30/CD30, AME-133v/CD20, HeFi-1/CD30,BMS-663513/CD137, Volociximab/anti-α5β1 integrin, GC1008/TGFβ,HCD122/CD40, Siplizumab/CD2, MORAb-003/Folate receptor alpha, CNTO328/IL-6, MDX-060/CD30, Ofatumumab/CD20, and SGN-33/CD33. It iscontemplated that one or more of these therapies may be employed withthe miRNA therapies described herein.

A number of different approaches for passive immunotherapy of cancerexist. They may be broadly categorized into the following: injection ofantibodies alone; injection of antibodies coupled to toxins orchemotherapeutic agents; injection of antibodies coupled to radioactiveisotopes; injection of anti-idiotype antibodies; and finally, purging oftumor cells in bone marrow.

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatment of thepresent invention, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised, and/or destroyed. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and microscopically controlled surgery(Mohs' surgery). It is further contemplated that the present inventionmay be used in conjunction with removal of superficial cancers,precancers, or incidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

Hormonal therapy may also be used in conjunction with the presentinvention or in combination with any other cancer therapy previouslydescribed. The use of hormones may be employed in the treatment ofcertain cancers such as breast, prostate, ovarian, or cervical cancer tolower the level or block the effects of certain hormones such astestosterone or estrogen. This treatment is often used in combinationwith at least one other cancer therapy as a treatment option or toreduce the risk of metastases.

IV. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Effect of Nucleotide Modifications in Passenger or ActiveStrands of miR-34a on Anti-Cell Proliferation Activity

The activity of a miRNA depends on both its ability to interact withproteins of the RNA-Induced Silencing Complex (RISC) and its ability tointeract with an mRNA target through hybridization. To determine whichnucleotides in a double-stranded miR-34a could be modified withoutdisrupting miRNA activity within cells, we used a series ofdouble-stranded miR-34a mimics with 2′oxygen-methyl (2′O-Me)-modifiednucleotides incorporated at two adjacent positions on the active strandor on the passenger strand of the double-stranded miRNA (Table 1). Inaddition to the 2′O-Me modifications, each passenger strand had a 5′-endmodification consisting of a 5′-amino C6 nucleotide (primary amine groupattached to a 6-carbon spacer).

TABLE 1Sequence and modification patterns of miR-34a mimics. Nucleotide locations of 2′O-Me-modified nucleotides are indicated as bold, italicized, and underlined.Position of 2′O-Me- 5′ Strand Modified Nucleotides ModificationSequence No. 4 Passenger AACAACCAGCUAAGACACUGCCA None 5′-amino C6Passenger

CAACCAGCUAAGACACUGCCA 1, 2 5′-amino C6 Passenger AA

ACCAGCUAAGACACUGCCA 3, 4 5′-amino C6 Passenger AACA

CAGCUAAGACACUGCCA 5, 6 5′-amino C6 Passenger AACAAC

GCUAAGACACUGCCA 7, 8 5′-amino C6 Passenger AACAACCA

UAAGACACUGCCA 9, 10 5′-amino C6 Passenger AACAACCAGC

AGACACUGCCA 11, 12 5′-amino C6 Passenger AACAACCAGCUA

ACACUGCCA 13, 14 5′-amino C6 Passenger AACAACCAGCUAAG

ACUGCCA 15, 16 5′-amino C6 Passenger AACAACCAGCUAAGAC

UGCCA 17, 18 5′-amino C6 Passenger AACAACCAGCUAAGACAC

CCA 19, 20 5′-amino C6 Passenger AACAACCAGCUAAGACACUG

A 21, 22 5′-amino C6 Passenger

ACAACCAGCUAAGACACUGCC

1, 23 5′-amino C6 Passenger

CAACCAGCUAAGACACUGC

1, 2, 22, 23 5′-amino C6 Sequence No. 2 Active UGGCAGUGUCUUAGCUGGUUGUUNone None Active

GCAGUGUCUUAGCUGGUUGUU 1, 2 None Active UG

AGUGUCUUAGCUGGUUGUU 3, 4 None Active UGGC

UGUCUUAGCUGGUUGUU 5, 6 None Active UGGCAG

UCUUAGCUGGUUGUU 7, 8 None Active UGGCAGUG

UUAGCUGGUUGUU 9, 10 None Active UGGCAGUGUC

AGCUGGUUGUU 11, 12 None Active UGGCAGUGUCUU

CUGGUUGUU 13, 14 None Active UGGCAGUGUCUUAG

GGUUGUU 15, 16 None Active UGGCAGUGUCUUAGC

GUUGUU 16, 17 None Active UGGCAGUGUCUUAGCUG

UGUU 18, 19 None Active UGGCAGUGUCUUAGCUGG

GUU 19, 20 None Active UGGCAGUGUCUUAGCUGGUU

21, 22, 23 None Active UG

AGUGUCUUAGCUG

UGUU 3, 4, 18, 19 None Active UG

AGUGUC

AGCUG

UGUU 3, 4, 11, 12, 18, 19 None Active UG

AGUGUC

AG

G

UGUU 3, 4, 11, 12, 15, 16, 18, 19 None Active UG

AGUGUC

AG

GG

GUU 3, 4, 11, 12, 15, 16, 19, 20 None

The inventors examined the effects of the oligonucleotide modificationson the activities of the miR-34a mimics. A synthetic, passenger strandoligonucleotide having a 5′-amino C6 modification and no 2′O-Memodifications was annealed to each of the synthetic, modified activestrand oligonucleotides (Table 1). A synthetic, unmodified active strandoligonucleotide was annealed to each of the synthetic, modifiedpassenger strand oligonucleotides (Table 1.) The lung cancer cell line(H460) was reverse transfected with the resultant double strandedoligonucleotides or with a negative control miRNA (Life Technologies,Inc./Ambion, Inc; Austin, Tex., USA; cat. no. AM17103) at finalconcentrations of 30 nM. Cell lines were transiently transfected usingLipofectamine 2000 (Life Technologies, Inc./Invitrogen Corp., Carlsbad,Calif., USA) according to the manufacturer's recommendations using thefollowing parameters: 5,000 cells per well in a 96 well plate, 0.1-0.2μl of Lipofectamine 2000 (cell line specific optimized), in 100 μl totalvolume of media. Cell viability assays were performed using theAlamarBlue® reagent (Invitrogen Corp.; Carlsbad, Calif., USA; cat. no.DAL1100) according to the manufacturer's protocol at days 3, 4, and 7following transfection. A fluorescent plate reader was used to measureaccumulation of resorufin (560 nm excitation, 590 nm emission) which isthe reduced substrate of AlamarBlue®, Resorufin accumulation isindicative of the number of viable cells per well. The relative numberof proliferating cells for each cell population transfected with adouble-stranded miR-34a having 2′O-Me modifications was calculated bydividing the fluorescence of cells transfected with the unmodifiedmiR-34a mimics by the fluorescence of cells transfected with thedouble-stranded 2′O-Me-modified miR-34a mimics and multiplying theresult by 100. Relative anti-proliferation values are shown in Table 2.

TABLE 2 Effect of 2′-O Me-modified nucleotides in miR-34a onproliferation of H460 lung cancer cells. Anti-cell proliferativeactivity of a synthetic, double-stranded miR-34a having no 2′O—Memodifications was set at 100%. Percentage anti-cell proliferation valuesgreater than 100 indicate anti-proliferative activity that is higherthan that of the unmodified miR-34a control. The indicated modificationswere in the passenger strand or active strand only. All passengerstrands had a 5′- amino C6 modification. Percentage Anti-CellProliferation Activity Modification Modification 2′O—Me-Modified inPassenger in Active Nucleotide Positions Strand Strand 1, 2 126 84 3, 4113 106 5, 6 89 89 7, 8 96 60 9, 10 115 60 11, 12 109 138 13, 14 134 12415, 16 74 110 16, 17 ND 86 17, 18 108 ND 18, 19 ND 65 19, 20 121 60 21,22 127 ND 21, 22, 23 ND 88 1, 23 98 ND 1, 2, 22, 23 96 ND 3, 4, 18, 19ND 58 3, 4, 11, 12, 18, 19 ND 58 3, 4, 11, 12, 15, 16, 18, 19 ND 53 3,4, 11, 12, 15, 16, 19, 20 ND 57 ND, not determined

As shown in Table 2, 2′O-Me modifications in the passenger strand atpositions 1+2; 3+4; 9+10; 11+12; 13+14; 17+18; 19+20; and 21+22 and inthe active strand at positions 3+4; 11+12; 13+14; and 15+16 resulted inincreased anti-proliferative activity over that observed for theunmodified control miR-34a. 2′O-Me modifications at positions 15+16 ofthe passenger strand and at positions 7+8; 9+10; 18+19; 19+20;3+4+18+19; 3+4+11+12+18+19; 3+4+11+12+15+16+18+19; and3+4+11+12+15+16+19+20 of the active strand resulted in substantiallyimpaired anti-proliferative activity when compared to the miR-34a mimichaving no 2′-O Me modifications. Modifications to other positions of thepassenger or active strands nominally affected the anti-proliferativeactivities of the mimics.

Example 2 Effect of Combined Nucleotide Modifications in Passenger andActive Strands of miR-34a on Anti-Cell Proliferation Activity

The inventors evaluated the anti-cell proliferation activity of miR-34amimics having modified nucleotides in both the passenger and activestrands. Various modified passenger and active strand oligonucleotideswere annealed to form miR-34a mimics with modifications on both strands.Lung cancer (H460), prostate cancer (PC3), and liver cancer (C3A) celllines were reverse transfected with the various mimics as well as with anegative control miRNA (Ambion, cat. no. AM17103) at finalconcentrations of 30 nM. Lipofectamine 2000 (Invitrogen) was usedaccording to the manufacturer's recommendations using the followingparameters: 5,000 cells per well in a 96 well plate, 0.1-0.2 μl ofLipofectamine 2000 (cell line optimized), in 100 μl total volume ofmedia. An AlamarBlue® assay (Invitrogen, cat. no. DAL1100) was performedon the cells according to the manufacturer's protocol, on day sixfollowing transfection. A fluorescent plate reader was used to measureaccumulation of resorufin (560 nm excitation, 590 nm emission) which isthe reduced substrate of AlamarBlue®. Resorufin fluorescence correlateswith the number of viable cells per well. The relative number ofproliferating cells for each transfected cell population was calculatedby dividing the fluorescence of cells transfected with the unmodifiedmiR-34a mimics by the fluorescence of cells transfected with thedouble-stranded 2′O-Me-modified miR-34a and multiplying the result by100. Results are shown in Table 3.

TABLE 3 Effects of nucleotide modifications in a double-stranded miR-34amimic on anti-cell proliferation activity of lung cancer cells (H460),prostate cancer cells (PC3), and liver cancer cells (C3A). Values forthe percentage of anti-cell proliferation activity represent an averageof that observed for all three cell lines. Values greater than 100indicate anti- proliferative activity that is higher with modifiedmiR-34a mimics than that observed with unmodified miR-34a. The indicatedmodifications were in the passenger strand, active strand, or bothstrands. All passenger strands had a 5′-amino C6 modification.Percentage Active Strand Passenger Strand Anti-Cell 2′O—Me-Modified2′O—Me-Modified Proliferation Nucleotide Positions Nucleotide PositionsActivity None None 100 None 1, 2, 20, 21, 22, 23 131 None 1, 2, 3, 4,22, 23 140 None 1, 2, 5, 6, 22, 23 131 None 1, 5, 6, 22, 23 133 None 1,2, 19, 20, 22, 23 131 11, 12, 15, 16 None 107 11, 12, 15, 16 1, 2, 20,21, 22, 23 152 11, 12, 15, 16 1, 2, 3, 4, 22, 23 138 11, 12, 15, 16 1,2, 5, 6, 22, 23 146 11, 12, 15, 16 1, 5, 6, 22, 23 146 11, 12, 15, 16 1,2, 19, 20, 22, 23 154 1, 2, 11, 12 None 99 1, 2, 11, 12 1, 2, 20, 21,22, 23 80 1, 2, 11, 12 1, 2, 3, 4, 22, 23 85 1, 2, 11, 12 1, 2, 5, 6,22, 23 66 1, 2, 11, 12 1, 5, 6, 22, 23 63 1, 2, 11, 12 1, 2, 19, 20, 22,23 75 3, 4, 11, 12 None 97 3, 4, 11, 12 1, 2, 20, 21, 22, 23 138 3, 4,11, 12 1, 2, 3, 4, 22, 23 132 3, 4, 11, 12 1, 2, 5, 6, 22, 23 122 3, 4,11, 12 1, 5, 6, 22, 23 122 3, 4, 11, 12 1, 2, 19, 20, 22, 23 130 3, 4,15, 16 None 90 3, 4, 15, 16 1, 2, 20, 21, 22, 23 143 3, 4, 15, 16 1, 2,3, 4, 22, 23 137 3, 4, 15, 16 1, 2, 5, 6, 22, 23 131 3, 4, 15, 16 1, 5,6, 22, 23 125 3, 4, 15, 16 1, 2, 19, 20, 22, 23 151 11, 12, 13, 14, 15,16, 17, 18 None 109 11, 12, 13, 14, 15, 16, 17, 18 1, 2, 20, 21, 22, 23159 11, 12, 13, 14, 15, 16, 17, 18 1, 2, 3, 4, 22, 23 138 11, 12, 13,14, 15, 16, 17, 18 1, 2, 5, 6, 22, 23 132 11, 12, 13, 14, 15, 16, 17, 181, 5, 6, 22, 23 139 11, 12, 13, 14, 15, 16, 17, 18 1, 2, 19, 20, 22, 23159

As shown in Table 3, all miR-34a mimics tested having 2′O-Memodifications only in the passenger strand have higher anti-cellproliferation activity than does the unmodified miR-34 mimic. Severalmimics having modifications in both active and passenger strands haveanti-cell proliferation activities that suggest synergistic effects ofthe modifications. For example, mimics having a 11,12,15,162′O-Me-modified active strand combined with 1,2,20,21,22,23;1,2,3,4,22,23; 1,2,5,6,22,23; 1,5,6,22,23; and 1,2,19,20,22,232′O-Me-modified passenger strands are considerably moreanti-proliferative than mimics with only one modified strand. A miR-34amimic with 2′O-Me modifications at positions 3,4,11,12 on the activestrand combined with 2′O-Me modifications at positions 1,2,20,21,22,23;1,2,3,4,22,23; 1,2,5,6,22,23; 1,5,6,22,23; and 1,2,19,20,22,23 on thepassenger strand demonstrated greater anti-proliferation activity thandid a mimic having only the active strand modification. These datasuggest that 2′O-Me modifications not only enhance theanti-proliferative activities of miR-34a mimics but that certaincombinations of modifications can be applied to significantly enhancethe activities of a miR-34a mimic.

Example 3 Nucleotide Modifications in Both Active and Passenger StrandsContribute to Stability of miRNA Mimics

Because a 2′OH is required for ribonucleases to cleave RNA molecules,incorporating 2′-modified nucleotides into RNA molecules can make themmore resistant to nuclease digestion. The inventors used an in vitrostability assay and purified RNase A (Ambion, cat. no. AM2270) tocompare the stabilities of the modified and unmodified miR-34a mimics.

miR-34a mimics were prepared by hybridizing complementaryoligonucleotides having 2′O-Me-modified nucleotides at various positionsand incubating the hybrids with of RNaseA (20 μg/ml) at 25° C. for 60min. Following the 60 min incubation dithiothreitol (DTT) was added to afinal concentration of 10 mM and the mixture was heated at 60° C. for 10mM to inactivate RNase activity. RNA was reverse transcribed withMMLV-RT (Invitrogen, cat. no. 28025-021) using the hsa-miR-34a TaqMan®MicroRNA assay RT primer (Applied Biosystems Inc.; cat. no. 4427975,assay ID 000425). qRT-PCR was performed on the cDNA using the TaqMan®MicroRNA assay with a primer specific for hsa-miR-34a and Platinum TaqPolymerase (Invitrogen, cat. no. 10966-083), in a 7900HT Fast Real-TimePCR System (Applied Biosystems). Table 4 shows the effects of nucleotidemodifications on the stability of various miR-34a mimics.

TABLE 4Effects of strand modifications on the stability of double-stranded miR-34a mimicsfollowing incubation with RNase A. Passenger and active strand sequences are shown. Bold,italicized, underlined letters indicate 2′O-Me-modified nucleotides. All passenger strands had a 5′-amino C6 modification. The active and passenger strands within each row were hybridized andincubated in an RNase A solution. The relative percentages of double-stranded mimics remainingafter RNase A treatment were calculated by determining the percentage of double-stranded mimicsremaining and dividing by the percentage of the unmodified double stranded mimic remaining.Values greater than 1.00 indicate modified mimics having more stability than the unmodified mimic.A value of 100 in the table would indicate that a modified miR-34a mimic was 100 times morestable than the unmodified miR-34a mimic. Relative amount of dsPassenger Active mimic after RNase P-ID SEQ. ID NO: 4 SEQ. ID NO: 2 A-IDtreatment 140 AACAACCAGCUAAGAC UGGCAGUGUCUUAGCUGG 150 1.00 ACUGCCA UUGUU210

CAACCAGCUAAGACA UGGCAGUGUCUUAGCUGG 150 4.55 CU

UUGUU 211

ACCAGCUAAGACA UGGCAGUGUCUUAGCUGG 150 2089.36 CUGC

UUGUU 215

CAACCAGCUAAGACA UGGCAGUGUCUUAGCUGG 150 4.49 C

C

UUGUU 210

CAACCAGCUAAGACA UGGCAGUGUC

AG

GG 200 2.98 CU

UUGUU 211

ACCAGCUAAGACA UGGCAGUGUC

AG

GG 200 666.52 CUGC

UUGUU 215

CAACCAGCUAAGACA UGGCAGUGUC

AG

GG 200 3.89 C

C

UUGUU 210

CAACCAGCUAAGACA UG

AGUGUCUUAG

GG 205 2.78 CU

UUGUU 211

ACCAGCUAAGACA UG

AGUGUCUUAG

GG 205 180.24 CUGC

UUGUU 215

CAACCAGCUAAGACA UG

AGUGUCUUAG

GG 205 3.81 C

C

UUGUU 210

CAACCAGCUAAGACA UGGCAGUGUC

206 2.52 CU

UUGUU 211

ACCAGCUAAGACA UGGCAGUGUC

206 593.44 CUGC

UUGUU 215

CAACCAGCUAAGACA UGGCAGUGUC

206 959.49 C

C

UUGUU A-ID, active strand ID number; P-ID, passenger strand ID number;ds, double-stranded.

The mimic having active and passenger strand combination A150/P211 isover 2000 times more stable than the unmodified mimic. Heightenedstability of mimics can profoundly improve pharmacodynamic propertiesduring therapy with miRNAs.

In the absence of these data, one might predict that the mimic with themost 2′O-Me-modified nucleotides would be the most stable, because ithas the fewest nuclease-sensitive sites. Surprisingly, in our assay, oneof the most modified mimics (A206/P210) was only slightly more stablethan the unmodified mimic. These data indicate that simply counting thenumber of 2′O-Me modifications is not an accurate reflection of or apredictable way for determining stability of modified double-strandedmiR-34a mimics.

Example 4 Nucleotide Modifications in Both Active and Passenger StrandsContribute to Activity of miRNA Mimics

The anti-proliferative activities of the miR-34a mimics were evaluatedand compared to the miR-34a mimic with no 2′O-Me modifications. Lungcancer cells (H460) were reverse transfected with the four differentdouble stranded miR-34a mimics and a negative control miRNA (Ambion,cat. no. AM17103) at final concentrations of 1, 3, and 10 nM.Lipofectamine 2000 (Invitrogen) was used according to the manufacturer'srecommendations using the following parameters: 5,000 cells per well ina 96 well plate, 0.1-0.2 μl of Lipofectamine 2000 (cell line specificoptimized), in 100 μl total volume of media. An AlamarBlue® assay(Invitrogen, cat. no. DAL1100) was performed on the cells at 3 dayspost-transfection, according to the manufacturer's protocol. Afluorescent plate reader was used to measure accumulation of resorufin(560 nm excitation, 590 nm emission) which is the reduced substrate ofAlamarBlue®. —Resorufin fluorescence correlates with the number ofviable cells per well. The relative percentage of viable cells for eachtransfected cell population was calculated by dividing the fluorescencefrom cells transfected with a given concentration of the miR-34a mimicby the fluorescence from cells transfected with the same concentrationof the negative control miRNA and multiplying the result by 100. Valuesless than 100% indicate that the miR-34a mimic reduced the number ofviable cells in the population relative to cell populations that weretransfected with the negative control miRNA. The results are shown inTable 5.

TABLE 5Effects of 2′-O Me modifications on the anti-cell proliferation activity of miR-34a mimicsfollowing transfection of H460 lung cancer cells. Passenger and active strand sequences areshown. Bold, italicized, underlined letters indicate 2′O-Me-modified nucleotides. The activeand the passenger strands within each row were hybridized and transfected into H460 cells atconcentrations shown. Passenger Active Percentage Viable Cells P-IDSEQ ID NO: 4) SEQ ID NO: 2 A-ID 3 nM 10 nM 30 nM 140 AACAACCAGCUAAGAUGGCAGUGUCUUAGC 150 54% 48% 43% CACUGCCA UGGUUGUU 210

CAACCAGCUAAGAC UGGCAGUGUCUUAGC 150 55% 51% 52% ACU

UGGUUGUU 211

ACCAGCUAAGAC UGGCAGUGUCUUAGC 150 50% 37% 39% ACUGC

UGGUUGUU 215

CAACCAGCUAAGAC UGGCAGUGUCUUAGC 150 59% 50% 50% AC

C

UGGUUGUU 210

CAACCAGCUAAGAC UGGCAGUGUC

AG

200 41% 36% 40% ACU

GGUUGUU 211

ACCAGCUAAGAC UGGCAGUGUC

AG

200 31% 25% 26% ACUGC

UGGUUGUU 215

CAACCAGCUAAGAC UGGCAGUGUC

AG

200 37% 28% 25% AC

C

GGUUGUU 210

CAACCAGCUAAGAC UG

AGUGUCUUAG

205 50% 39% 42% ACU

GGUUGUU 211

ACCAGCUAAGAC UG

AGUGUCUUAG

205 45% 32% 34% ACUGC

GGUUGUU 215

CAACCAGCUAAGAC UG

AGUGUCUUAG

205 52% 42% 43% AC

C

GGUUGUU 210

CAACCAGCUAAGAC UGGCAGUGUC

206 36% 30% 31% ACU

UUGUU 211

ACCAGCUAAGA UGGCAGUGUC

206 30% 23% 24% ACUGC

UUGUU 215

CAACCAGCUAAGAC UGGCAGUGUC

206 35% 27% 30% AC

C

GUUGUU A-ID, active strand ID number; P-ID, passenger strand ID number.Percentage viable cells is the percentage of cells that remain viablefollowing transfection with the miR-34a mimic. Values for the negativecontrol miRNA were set a 100%. All passenger strands had a 5′-amino C6modification.

The 2′-O Me modified mimic pairs all demonstrated enhancednuclease-stability relative to the unmodified mimic (Table 4), andseveral of the dual strand modified mimics also exhibited increasedanti-proliferative activity (Table 5). Most interesting were the dualstrand modified mir-34a mimics that had similar anti-proliferativeactivities as the standard miR-34a mimic when used at one-third the dosesince it indicated that the modified miR-34a mimics were approximatelythree times more active than the standard miR-34a mimic. As with thestability data, it was not possible to predict which modificationcombinations would produce the most active miRNA mimics.

Example 5 Gene Regulation by Modified miR-34a Mimics

miRNAs function as guide sequences for the RNA-Induced Silencing Complex(RISC) regulation of mRNA translation. After entering the RISC, a miRNAmimic can alter the mRNA profiles of transfected cells by: (1) inducingRISC to cleave an mRNA that is bound to the miRNA, (2) altering thehalf-life of a bound mRNA by preventing it from interacting withribosomes, and/or (3) causing changes in amounts of mRNAs that areregulated by genes that themselves are regulated by the miRNA mimic.

To address whether the modified miR-34a mimics have the same effects onmRNA expression that an unmodified miR-34a mimic has, we used mRNAarrays to profile gene transcription in H460 lung cancer cellstransfected with a negative control miRNA (Ambion, cat. no. AM17111), anunmodified miR-34a mimic (P140/A150), or five different modified miR-34amimics (P210/A150, P211/A150, P215/A150, P211/A200, and P211/A206)(Table 5). miRNA mimics at 3 nM or 10 nM were complexed with 0.2 μl ofLipofectamine 2000 and added to H460 cells at 5,000 cells per well in a96 well plate, in 100 μl total volume of RPMI media. Cells wereharvested at 3 days post transfection, and total RNA was extracted usingthe MirVana™ PARIS™ Kit (Ambion, cat. no. AM1556) following themanufacturer's recommended protocol.

mRNA array analyses were performed by Asuragen, Inc. (Austin, Tex.,USA), according to the company's standard operating procedures. Usingthe MessageAmp™ 11-96 aRNA Amplification Kit (Ambion, cat. no. 1819),200 ng of input total RNA were labeled with biotin. cRNA yields werequantified using an Agilent 2100 Bioanalyzer capillary electrophoresisinstrument (Agilent Technologies, Inc.). Labeled target was hybridizedto Affymetrix mRNA arrays (Human HG-U133A 2.0 arrays) using themanufacturer's recommendations and the following parameters.Hybridizations were carried out at 45° C. for 16 hr in an AffymetrixModel 640 hybridization oven. Arrays were washed and stained on anAffymetrix FS450 Fluidics station, running the wash scriptMidi_euk2v3_(—)450. The arrays were scanned on an Affymetrix GeneChipScanner 3000. Summaries of the image signal data, group mean values,p-values with significance flags, log ratios and gene annotations forevery gene on the array were generated using the Affymetrix StatisticalAlgorithm MAS 5.0 (GCOS v1.3). Data were reported containing theAffymetrix data and result files (cabinet file) and containing theprimary image and processed cell intensities of the arrays (.cel).

The mRNA array profiles for the various samples were compared todetermine their similarities. Pearson product-moment correlationcoefficients between the samples (complete probe set) were calculatedand are shown in Tables 6 and 7. Correlation coefficients for allsamples were observed to be greater than 0.987.

TABLE 6 Pearson product-moment correlation coefficients following arrayanalysis of gene expression after transfection of lung cancer cells with3 nM of the indicated miRNA mimic. Sequences and 2′-O Me modificationsof the P—passenger and A—active strands are shown in Table 5. Allpassenger strands had a 5′-amino C6 modification. P140/A150 P210/A150P211/A150 P215/A150 P211/A200 P211/A206 1.000 0.9974 0.9936 0.99710.9966 0.9970 P140/A150 1.000 0.9936 0.9974 0.9969 0.9966 P210/A1501.000 0.9953 0.9937 0.9943 P211/A150 1.000 0.9968 0.9965 P215/A150 1.0000.9975 P211/A200 1.000 P211/A206

TABLE 7 Pearson product-moment correlation coefficients following arrayanalysis of gene expression after transfection of lung cancer cells with10 nM of the indicated miRNA mimic. Sequences and 2′-O Me modificationsof the P—passenger and A—active strands are shown in Table 5. Allpassenger strands had a 5′-amino C6 modification. P140/A150 P210/A150P211/A150 P215/A150 P211/A200 P211/A206 1.000 0.9959 0.9962 0.99520.9932 0.9912 P140/A150 1.000 0.9959 0.9956 0.9915 0.9889 P210/A1501.000 0.9955 0.9910 0.9885 P211/A150 1.000 0.9906 0.9870 P215/A150 1.0000.9973 P211/A200 1.000 P211/A206

Limiting analysis to those mRNAs whose expression levels were altered atleast two-fold by a minimum of two miR-34a mimics, a strong correlationwas observed between cells transfected with the 2′-O Me modified miR-34amimics and the unmodified miR-34a mimic (Table 8). These data revealthat the target specificities of the modified miR-34a mimics are similarto the unmodified miR-34a mimic.

TABLE 8 Pearson product-moment correlation coefficients following arrayanalysis of gene expression altered at least two-fold after transfectionof lung cancer cells with 10 nM or 3 nM of the miR-34a mimic having nomodified nucleotides (P140/A150) and miR-34a mimics containing 2′-O Memodifications (P210/A150, P211/A150, P215/A150, P211/A200, P211/A206).Sequences and 2′-O Me modifications of the P-passenger and A-activestrands are shown in Table 5. All passenger strands had a 5′-amino C6modification. P210/ A150 P211/A150 P215/A150 P211/A200 P211/A206 10 nM0.9537 0.9543 0.9166 0.9409 0.90880 P140/ A150 3 nM 0.9591 0.9206 0.95920.9135 0.9199 P140/ A150

In addition to global expression profiles, we compared the activities of2′O-Me-modified and unmodified miR-34a mimics on known miR-34a targetgenes. Array data revealed that levels of two direct mRNA targets ofmiR-34a, MET and SIRT1, were significantly reduced in all of the cellpopulations treated with 10 nM of miR-34a mimics when compared to cellstransfected with a negative control miRNA (Table 9).

TABLE 9 MET or SIRT1 mRNA levels following transfection of H460 cellswith the indicated miR-34a mimic at a concentration of 3 nM or 10 nM.Values represent percentage expression compared to that observedfollowing transfection of cells with a negative control miRNA (100%).Sequences and 2′-O Me modifications of the P-passenger and A-activestrands are shown in Table 5. All passenger strands had a 5′-amino C6modification. Percentage Expression vs. Negative Control miRNA MET SIRT1miR-34a Mimic 3 nM 10 nM 3 nM 10 nM P140/A150 90.38 76.71 80.20 72.44P210/A150 84.17 62.16 66.88 52.76 P211/A150 105.83 64.36 102.69 51.56P215/A150 83.05 69.29 72.91 53.56 P211/A200 85.54 80.39 81.46 77.03P211/A206 93.09 84.75 94.10 84.44

Example 7 Effect of Nucleotide Modifications in Passenger or ActiveStrands of miR-34C on Anti-Cell Proliferation Activity

The activity of a miRNA depends on both its ability to interact withproteins of the RNA-Induced Silencing Complex (RISC) and its ability tointeract with an mRNA target through hybridization. To determine whichnucleotides in a double-stranded RNA mimic of mir-34c could be modifiedwithout disrupting miRNA activity within cells, we used a series ofdouble-stranded mir-34c mimics with 2′O-Me-modified nucleotidesincorporated at two adjacent positions on the active strand or on thepassenger strand of the double-stranded miRNA (Table 10). In addition tothe 2′O-Me modifications, each passenger strand had a 5′-amino C6modification.

TABLE 10Sequence and modification patterns of mir-34c mimics. Nucleotide locations of 2′O-Me-modified nucleotides are indicated as bold, italicized, and underlined.Sequence Passenger is SEQ ID NO: 6 Position of 2′O-Me 5′ StrandActive is SEQ ID NO: 5 Modified Nucleotides Modification PassengerGCAAUCAGCUAACUACACUGCCU None 5′-amino C6 Passenger

AAUCAGCUAACUACACUGCCU 1, 2 5′-amino C6 Passenger GC

UCAGCUAACUACACUGCCU 3, 4 5′-amino C6 Passenger GCAA

AGCUAACUACACUGCCU 5, 6 5′-amino C6 Passenger GCAAUC

CUAACUACACUGCCU 7, 8 5′-amino C6 Passenger GCAAUCAG

AACUACACUGCCU 9, 10 5′-amino C6 Passenger GCAAUCAGCU

CUACACUGCCU 11, 12 5′-amino C6 Passenger GCAAUCAGCUAA

ACACUGCCU 13, 14 5′-amino C6 Passenger GCAAUCAGCUAACU

ACUGCCU 15, 16 5′-amino C6 Passenger GCAAUCAGCUAACUAC

UGCCU 17, 18 5′-amino C6 Passenger GCAAUCAGCUAACUACAC

CCU 19, 20 5′-amino C6 Passenger

AAUCAGCUAACUACACUGC

1, 2, 22, 23 5′-amino C6 Passenger

AUCAGCUAACUACACUG

1, 2, 3, 21, 22, 23 5′-amino C6 Active AGGCAGUGUAGUUAGCUGAUUGC None NoneActive

GCAGUGUAGUUAGCUGAUUGC 1, 2 None Active AG

AGUGUAGUUAGCUGAUUGC 3, 4 None Active AGGC

UGUAGUUAGCUGAUUGC 5, 6 None Active AGGCAG

UAGUUAGCUGAUUGC 7, 8 None Active AGGCAGUG

GUUAGCUGAUUGC 9, 10 None Active AGGCAGUGUA

UAGCUGAUUGC 11, 12 None Active AGGCAGUGUAGU

GCUGAUUGC 13, 14 None Active AGGCAGUGUAGUUA

UGAUUGC 15, 16 None Active AGGCAGUGUAGUUAGC

AUUGC 17, 18 None Active AGGCAGUGUAGUUAGCUG

UGC 19, 20 None Active

GCAGUGUAGUUAGCUGAUU

1, 2, 22, 23 None Active

CAGUGUAGUUAGCUGAU

1, 2, 3, 21, 22, 23 None

The inventors examined the effects of the oligonucleotide modificationson the activities of the mir-34c mimics. A synthetic, passenger strandoligonucleotide having a 5′-amino C6 modification and no 2′O-Memodifications was annealed to each of the synthetic, modified activestrand oligonucleotides (Tables 10, 11). A synthetic, unmodified activestrand oligonucleotide was annealed to each of the synthetic, modifiedpassenger strand oligonucleotides (Tables 10, 11). The lung cancer cellline (H460) was reverse transfected with the resultant double strandedoligonucleotides or with a negative control miRNA (Life Technologies,Inc./Ambion, Inc; Austin, Tex., USA; cat. no. AM17103) at finalconcentrations of 30 nM. Cell lines were transiently transfected usingLipofectamine 2000 (Life Technologies, Inc./Invitrogen Corp., Carlsbad,Calif., USA) according to the manufacturer's recommendations using thefollowing parameters: 5,000 cells per well in a 96 well plate, 0.1-0.2μl of Lipofectamine 2000 (cell line specific optimized), in 100 μl totalvolume of media. Cell viability assays were performed using theAlamarBlue® reagent (Invitrogen Corp.; Carlsbad, Calif., USA; cat. no.DAL1100) according to the manufacturer's protocol at days 3, 4, and 7following transfection. A fluorescent plate reader was used to measureaccumulation of resorufin (560 nm excitation, 590 nm emission) which isthe reduced substrate of AlamarBlue®. Resorufin accumulation isindicative of the number of viable cells per well. The relative numberof proliferating cells for each cell population transfected with adouble-stranded mir-34c having 2′O-Me modifications was calculated bydividing the fluorescence of cells transfected with the unmodifiedmir-34c mimics by the fluorescence of cells transfected with2′O-Me-modified mir-34c mimics and multiplying the result by 100.Relative anti-proliferation values are shown in Table 11.

TABLE 11 Effects of nucleotide modifications in double-stranded miR-34cmimics on proliferation of lung cancer cells (H460). Percentageanti-cell proliferation values greater than 100 indicateanti-proliferative activity that is higher than that observed with amiR-34c control having no 2′-O—Me modifications in either strand. Theindicated modifications were in the passenger strand or active strandonly. All passenger strands had a 5′-amino C6 modification. 2′O—MeModified Percentage Anti-Cell Nucleotide Positions ProliferationActivity Passenger Strand Modified None 100 1, 2 136 3, 4 171 5, 6 84 7,8 127 9, 10 132 11, 12 143 13, 14 110 15, 16 69 17, 18 97 19, 20 106 1,2, 22, 23 143 1, 2, 3, 21, 22, 23 114 Active Strand Modified None 100 1,2 87 3, 4 98 5, 6 99 7, 8 79 9, 10 82 11, 12 132 13, 14 102 15, 16 9917, 18 99 19, 20 78 1, 2, 22, 23 89 1, 2, 3, 21, 22, 23 75

As shown in Table 11, 2′O-Me modifications in the passenger strand atpositions 1,2; 3,4; 5,6, 7,8; 9,10; 11,12; 13,14; 17,18; 19,20;1,2,22,23; and 1,2,3,21,22,23 and in the active strand at positions 1,2;3,4; 5,6; 9,10; 11,12; 13,14; 15,16; 17,18; and 1,2,22,23 resulted insimilar or increased anti-proliferative activity over that observed forthe unmodified control mir-34c. 2′O-Me modifications at positions 15+16of the passenger strand and at positions 7,8; 19,20; and 1,2,3,21,22,23of the active strand resulted in substantially impairedanti-proliferative activity when compared with the mir-34c mimic havingno 2′-O Me modifications.

Example 8 Effect of Combined Nucleotide Modifications in Passenger andActive Strands of miR-34C on Anti-Cell Proliferative Activity

The inventors evaluated the anti-cell proliferative activities ofmir-34c mimics having modified nucleotides in both the passenger andactive strands. Various modified passenger and active strandoligonucleotides were annealed to form mir-34c mimics with modificationson both strands. H460 lung cancer cells were reverse transfected withthe various mimics or with a negative control miRNA (Ambion, cat. no.AM17103) at final concentrations of 30 nM. Lipofectamine 2000(Invitrogen) was used according to the manufacturer's recommendationsusing the following parameters: 5,000 cells per well in a 96 well plate,0.1-0.2 μl of Lipofectamine 2000 (cell line optimized), in 100 μl totalvolume of media. An AlamarBlue® assay (Invitrogen, cat. no. DAL1100) wasperformed on the cells according to the manufacturer's protocol, on thesixth day after transfection. A fluorescent plate reader was used tomeasure accumulation of resorufin (560 nm excitation, 590 nm emission)which is the reduced substrate of alamarBlue. Resorufin fluorescencecorrelates with the number of viable cells per well. The relative numberof proliferating cells for each transfected cell population wascalculated by dividing the fluorescence of cells transfected with themodified mir-34c mimics by the fluorescence of cells transfected with2′O-Me-modified mir-34c mimics and multiplying the result by 100.Results are shown in Table 12.

TABLE 12 Effects of nucleotide modifications in double-stranded miR-34cmimics on proliferation of lung cancer cells (H460). Percentageanti-cell proliferation values greater than 100 indicateanti-proliferative activity that is higher than that observed with amiR-34c control having no 2′O—Me modifications in either strand. Theindicated modifications were in the passenger strand or active strandonly. All passenger strands had a 5′-amino C6 modification. PercentageActive Strand Passenger Strand Anti-Cell 2′O—Me Modified 2′O—Me ModifiedProliferation Nucleotide Positions Nucleotide Positions Activity NoneNone 100 None 17, 18 97 None 1, 2, 3, 21, 22, 23 89 None 1, 2, 3, 17,18, 21, 86 22, 23 None 1, 2, 3, 11, 12, 13, 14, 95 17, 18, 21, 22, 23 3,4, 17, 18 None 109 3, 4, 17, 18 17, 18 99 3, 4, 17, 18 1, 2, 3, 21, 22,23 97 3, 4, 17, 18 1, 2, 3, 17, 18, 21, 94 22, 23 3, 4, 17, 18 1, 2, 3,11, 12, 13, 14, 101 17, 18, 21, 22, 23 11, 12, 17, 18 None 138 11, 12,17, 18 17, 18 157 11, 12, 17, 18 1, 2, 3, 21, 22, 23 142 11, 12, 17, 181, 2, 3, 17, 18, 21, 135 22, 23 11, 12, 17, 18 1, 2, 3, 11, 12, 13, 14,158 17, 18, 21, 22, 23 3, 4, 11, 12, 13, 14, 15, 16, 17, 18 None 143 3,4, 11, 12, 13, 14, 15, 16, 17, 18 17, 18 158 3, 4, 11, 12, 13, 14, 15,16, 17, 18 1, 2, 3, 21, 22, 23 139 3, 4, 11, 12, 13, 14, 15, 16, 17, 181, 2, 3, 17, 18, 21, 153 22, 23 3, 4, 11, 12, 13, 14, 15, 16, 17, 18 1,2, 3, 11, 12, 13, 14, 159 17, 18, 21, 22, 23

As shown in Table 12, all mimics having 2′O-Me modifications in thepassenger strand have at least similar anti-cell proliferation activityto that of the unmodified mir-34c mimic. Several mimics havingmodifications in both active and passenger strands have anti-cellproliferation activities that suggest synergistic effects of themodifications. For example, mimics having the 11,12,17,182′O-Me-modified active strand combined with the 17,18; 1,2,3,21,22,23;or 1,2,3,11,12,13,14,17,18,21,22,23 2′O-Me-modified passenger strandsare more anti-proliferative than mimics with only one modified strand.These data suggest that 2′O-Me modifications not only enhance theanti-proliferative activities of mir-34c mimics but that certaincombinations of modifications can be applied to significantly enhancethe activities of a mir-34c mimic.

Example 9 Nucleotide Modifications in Both Active and Passenger StrandsContribute to Stability of miRNA Mimics

Because a 2′OH is required for ribonucleases to cleave RNA molecules,incorporating 2′-modified nucleotides into RNA molecules can make themmore resistant to nuclease digestion. The inventors used an in vitrostability assay and purified RNase A (Ambion, cat. no. AM2270) tocompare the stabilities of the modified and unmodified mir-34c mimics.

mir-34c mimics were prepared by hybridizing complementaryoligonucleotides having 2′O-Me-modified nucleotides at various positionsand incubating the hybrids with RNaseA (20 μg/ml) at 25° C. for 120 min.Following the 120 min incubation, dithiothreitol (DTT) was added to afinal concentration of 10 mM, and the mixture was heated at 60° C. for10 min to inactivate RNase activity. RNA was reverse transcribed withMMLV-RT (Invitrogen, cat. no. 28025-021) using the hsa-mir-34c TaqMan®MicroRNA assay RT primer (Applied Biosystems Inc.; cat. no. 4427975,assay ID 000428). qRT-PCR was performed on the cDNA using the TaqMan®MicroRNA assay with a primer specific for hsa-mir-34c and Platinum TaqPolymerase (Invitrogen, cat. no. 10966-083), in a 7900HT Fast Real-TimePCR System (Applied Biosystems). Table 13 shows the effects ofnucleotide modifications on the stability of various mir-34c mimics.

TABLE 13Effects of strand modifications on the stability of double-stranded mir-34c mimicsfollowing incubation with RNase A. Passenger and active strand sequences are shown. Bold,italicized, and underlined letters indicate 2′O-Me-modified nucleotides. All passenger strandshad a 5′-amino C6 modification. The active and passenger strands within each row were hybridizedtogether and then incubated in an RNase A solution. The relative percentages of double-strandedmimics remaining after RNase A treatment were calculated by determining the percentage ofdouble-stranded mimics remaining and dividing by the percentage of the unmodified doublestranded mimic remaining. Values greater than 1.00 indicate modified mimics having more stabilitythan the unmodified mimic. A value of 100 in the table would indicate that a modified mir-34cmimic was 100 times more stable than the unmodified mir-34c mimic.Relative amount of ds mir-34c mimic after P-ID Passenger (SEQ ID NO: 6)Active (SEQ ID NO: 5) A-ID RNase treatment 121 GCAAUCAGCUAACUACAGGCAGUGUAGUUAG 130 1.0 ACUGCCU CUGAUUGC 125

AUCAGCUAACUAC AGGCAGUGUAGUUAG 130 2327.9

UG

CUGAUUGC 126

AUCAGCU

AC AGGCAGUGUAGUUAG 130 1380.1 ACUG

CUGAUUGC 129

AUCAGCU

AC AGGCAGUGUAGUUAG 130 1972.5

UG

CUGAUUGC 129

AUCAGCU

AC AGGCAGUGUA

UAG 133 2700.2

UG

C

AUUGC A-ID, active strand ID number; P-ID, passenger strand ID number.

The mimic having active and passenger strand combination A133/P129 isover 2700 times more stable than the unmodified mimic. Heightenedstability of mimics can profoundly improve pharmacodynamic propertiesduring therapy with miRNAs.

In the absence of these data, one might predict that the mimic with themost 2′O-Me-modified nucleotides would be the most stable because it hasthe fewest nuclease-sensitive sites. Surprisingly, in our assay, mimiccombination A130/P126, having ten 2′O-Me-modified nucleotides was notobserved to be more stable than the A130/P125 mimic, having eight2′O-Me-modified nucleotides. These data indicate that simply countingthe number of 2′O-Me modifications is not an accurate reflection of or apredictable way for determining stability of modified double-strandedmir-34c mimics.

Example 10 Nucleotide Modifications in Both Active and Passenger StrandsContribute to Activity of miRNA Mimics

The anti-proliferative activities of four of the most nuclease-resistantmir-34c mimics were evaluated and compared to the mir-34c mimic with no2′O-Me modifications. Lung cancer cells (H460) were reverse transfectedwith the four different double stranded mir-34c mimics and a negativecontrol miRNA (Ambion, cat. no. AM17103) at final concentrations of 3,10, and 30 nM. Lipofectamine 2000 (Invitrogen) was used according to themanufacturer's recommendations using the following parameters: 5,000cells per well in a 96 well plate, 0.1-0.2 μl of Lipofectamine 2000(cell line specific optimized), in 100 μl total volume of media. AnalamarBlue® assay (Invitrogen, cat. no. DAL1100) was performed on thecells at 3 days post-transfection, according to the manufacturer'sprotocol. A fluorescent plate reader was used to measure accumulation ofresorufin (560 nm excitation, 590 nm emission) which is the reducedsubstrate of alamarBlue®. Resorufin fluorescence correlates with thenumber of viable cells per well. The relative percentage of viable cellsfor each transfected cell population was calculated by dividing thefluorescence from cells transfected with a given concentration of themir-34c mimic by the fluorescence from cells transfected with the sameconcentration of the negative control miRNA and multiplying the resultby 100. Values less than 100% indicate that the mir-34c mimic reducedthe number of viable cells in the population relative to cellpopulations that were transfected with the negative control miRNA. Theresults are shown in Table 14.

TABLE 14Effects of 2′O-Me modifications on the anti-cell proliferation activity of mir-34c mimicsfollowing transfection of H460 lung cancer cells. Passenger and active strand sequences are shown.Bold, italicized, and underlined letters indicate 2′O-Me-modified nucleotides. The active andpassenger strands within each row were hybridized and transfected into H460 cells at the finalconcentrations shown. Percentage Viable Cells P-IDPassenger (SEQ ID NO: 6) Active (SEQ ID NO: 5) A-ID 3 nM 10 nM 30 nM 121GCAAUCAGCUAACUAC AGGCAGUGUAGUUAG 130 85% 60% 59% ACUGCCU CUGAUUGC 125

AUCAGCUAACUAC AGGCAGUGUAGUUAG 130 86% 66% 71%

UG

CUGAUUGC 126

AUCAGCU

AC AGGCAGUGUAGUUAG 130 86% 65% 60% ACUG

CUGAUUGC 129

AUCAGCU

AC AGGCAGUGUAGUUAG 130 88% 71% 71%

UG

CUGAUUGC 129

AUCAGCU

AC AGGCAGUGUA

UAG 133 56% 42% 40%

UG

C

AUUGC A-ID, active strand ID number; P-ID, passenger strand ID number.Percentage viable cells represents the percentage of cells that remainviable following transfection with the mir-34c mimic. Values for thenegative control miRNA were set a 100%. All passenger strands had a5′-amino C6 modification.

All mimic pairs demonstrated enhanced nuclease-stability relative to theunmodified mimic (Table 13) and the mimic with 2′O-Me modifications inboth strands also exhibited increased anti-proliferative activity (Table14). The mimic with modifications in both strands displayedanti-proliferative activity similar to that of the control mimic whenused at one-tenth the dose.

Example 11 Gene Regulation by Modified miR-34C Mimics

miRNAs function as guide sequences for the RNA-Induced Silencing Complex(RISC) to regulate the translation of mRNAs. After entering the RISC, amiRNA mimic can alter the mRNA profiles of transfected cells by: (1)inducing RISC to cleave an mRNA that is bound to the miRNA, (2) alteringthe half-life of a bound mRNA by preventing it from interacting withribosomes, and/or (3) causing changes in amounts of mRNAs that areregulated by genes that themselves are regulated by the miRNA mimic.

To address whether the modified mir-34c mimics have the same effects onmRNA expression that an unmodified mir-34c mimic has, we used mRNAarrays to profile gene transcription in H460 lung cancer cellstransfected with two different negative control miRNAs (Ambion, cat. no.AM17111; Ambion, cat. no. AM17103), an unmodified mir-34c mimic(P121/A130), or four different modified mir-34c mimics (P125/A130,P126/A130, P129/A130, and P129/A133) (Table 14). miRNA mimics at 3, 10,or 30 nM were complexed with 0.2 μl of Lipofectamine 2000 and added toH460 cells at 5,000 per well in a 96 well plate, in 100 μl total volumeof RPMI media. Cells were harvested at 3 days post transfection, andtotal RNA was extracted using the MirVana™ PARIS™ Kit (Ambion, cat. no.AM1556) following the manufacturer's recommended protocol.

mRNA array analyses were performed by Asuragen, Inc. (Austin, Tex.,USA), according to the company's standard operating procedures. Usingthe MessageAmp™ 11-96 aRNA Amplification Kit (Ambion, cat. no. 1819),200 ng of input total RNA were labeled with biotin. cRNA yields werequantified using an Agilent 2100 Bioanalyzer capillary electrophoresisinstrument (Agilent Technologies, Inc.). Labeled target was hybridizedto Affymetrix mRNA arrays (Human HG-U133A 2.0 arrays) using themanufacturer's recommendations and the following parameters.Hybridizations were carried out at 45° C. for 16 hr in an AffymetrixModel 640 hybridization oven. Arrays were washed and stained on anAffymetrix FS450 Fluidics station, running the wash scriptMidi_euk2v3_(—)450. The arrays were scanned on an Affymetrix GeneChipScanner 3000. Summaries of the image signal data, group mean values,p-values with significance flags, log ratios and gene annotations forevery gene on the array were generated using the Affymetrix StatisticalAlgorithm MAS 5.0 (GCOS v1.3). Data were reported containing theAffymetrix data and result files (cabinet file) and containing theprimary image and processed cell intensities of the arrays (.cel).

The mRNA array profiles for the various samples were compared todetermine their similarities. Pearson product-moment correlationcoefficients between the samples (complete probe set) were calculatedand are shown in Tables 15, 16, and 17. Correlation coefficients for allsamples were observed to be greater than 0.9934.

TABLE 15 Pearson product-moment correlation coefficients following arrayanalysis of gene expression after transfection of lung cancer cells with3 nM of the indicated miRNA mimic. Sequences and 2′O—Me modifications ofthe passenger (P) and active (A) strands are shown in Table 14. Allpassenger strands had a 5′-amino C6 modification. P121/ P125/ A130 A130P126/A130 P129/A130 P129/A133 1.000 0.9984 0.9987 0.9982 0.9968P121/A130 1.000 0.9982 0.9979 0.9978 P125/A130 1.000 0.9986 0.9969P126/A130 1.000 0.9969 P129/A130 1.000 P129/A133

TABLE 16 Pearson product-moment correlation coefficients following arrayanalysis of gene expression after transfection of lung cancer cells with10 nM of the indicated miRNA mimic. Sequences and 2′O—Me modificationsof the passenger (P) and active (A) strands are shown in Table 14. Allpassenger strands had a 5′-amino C6 modification. P121/ P125/ A130 A130P126/A130 P129/A130 P129/A133 1.000 0.9972 0.9980 0.9962 0.9968P121/A130 1.000 0.9973 0.9952 0.9978 P125/A130 1.000 0.9968 0.9965P126/A130 1.000 0.9934 P129/A130 1.000 P129/A133

TABLE 17 Pearson product-moment correlation coefficients following arrayanalysis of gene expression after transfection of lung cancer cells with30 nM of the indicated miRNA mimic. Sequences and 2′O—Me modificationsof the passenger (P) and active (A) strands are shown in Table 14. Allpassenger strands had a 5′-amino C6 modification. P121/ P125/ A130 A130P126/A130 P129/A130 P129/A133 1.000 0.9961 0.9963 0.9982 0.9955P121/A130 1.000 0.9965 0.9962 0.9971 P125/A130 1.000 0.9973 0.9960P126/A130 1.000 0.9956 P129/A130 1.000 P129/A133

Limiting analysis to those mRNAs whose expression levels relative to thenegative control were altered at least two-fold by a minimum of twomir-34c mimics, strong correlation was observed between cellstransfected with the 2′O-Me modified mir-34c mimics and the unmodifiedmir-34c mimic (Table 18). These data reveal that the targetspecificities of the modified mir-34c mimics are similar to theunmodified mir-34c mimic.

TABLE 18 Pearson product-moment correlation coefficients following arrayanalysis of gene expression altered at least two-fold after transfectionof lung cancer cells with 30, 10, or 3 nM of the mir-34c mimic having nomodified nucleotides (P121/A130) and mir-34c mimics containing 2′O—Memodifications (P125/A130, P126/A130, P129/A133, P129/A133). Sequencesand 2′O—Me modifications of the passenger (P) and active (A) strands areshown in Table 14. All passenger strands had a 5′-amino C6 modification.P125/A130 P126/A130 P129/A130 P129/A133 30 nM 0.8853 0.9307 0.95620.8768 P121/A130 10 nM 0.8828 0.9456 0.8992 0.9008 P121/A130 3 nM 0.93500.9459 0.9318 0.8629 P121/A130

In addition to global expression profiles, we compared the activity of2′O-Me-modified and unmodified mir-34c mimics on predicted mir-34ctarget genes. Array data revealed that levels of two predicted mRNAtargets of mir-34c, NR4A2 and SYT1, were significantly reduced in themir-34c-treated cell populations when compared to cells transfected witha negative control miRNA (Table 19).

TABLE 19 NR4A2 or SYT1 mRNA levels following transfection of H460 cellswith the indicated mir-34c mimic at a concentration of 3, 10, or 30 nM.Values represent percentage expression compared to that observedfollowing transfection of cells with a negative control miRNA (100%).Sequences and 2′O-Me modifications of the passenger (P) and active (A)strands are shown in Table 14. All passenger strands had a 5′-amino C6modification. Percentage Expression vs. Negative Control miRNA NR4A2SYT1 miR-34c Mimic 3 nM 10 nM 30 nM 3 nM 10 nM 30 nM P121/A130 67% 34%39% 89% 69% 71% P125/A130 48% 28% 19% 86% 54% 52% P126/A130 73% 46% 29%90% 76% 74% P129/A130 58% 33% 39% 93% 70% 85% P129/A133 13% 17% 30% 56%36% 42%

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. No. 3,687,808-   U.S. Pat. No. 3,832,253-   U.S. Pat. No. 3,854,480-   U.S. Pat. No. 4,452,775-   U.S. Pat. No. 4,659,774-   U.S. Pat. No. 4,667,013-   U.S. Pat. No. 4,675,189-   U.S. Pat. No. 4,682,195-   U.S. Pat. No. 4,683,202-   U.S. Pat. No. 4,704,362-   U.S. Pat. No. 4,748,034-   U.S. Pat. No. 4,816,571-   U.S. Pat. No. 4,870,287-   U.S. Pat. No. 4,959,463-   U.S. Pat. No. 4,981,957-   U.S. Pat. No. 5,075,109-   U.S. Pat. No. 5,118,800-   U.S. Pat. No. 5,133,974-   U.S. Pat. No. 5,141,813-   U.S. Pat. No. 5,221,619-   U.S. Pat. No. 5,239,660-   U.S. Pat. No. 5,264,566-   U.S. Pat. No. 5,319,080-   U.S. Pat. No. 5,359,044-   U.S. Pat. No. 5,393,878-   U.S. Pat. No. 5,399,363-   U.S. Pat. No. 5,407,686-   U.S. Pat. No. 5,428,148-   U.S. Pat. No. 5,446,137-   U.S. Pat. No. 5,466,468-   U.S. Pat. No. 5,466,786-   U.S. Pat. No. 5,514,785-   U.S. Pat. No. 5,519,134-   U.S. Pat. No. 5,543,158-   U.S. Pat. No. 5,554,744-   U.S. Pat. No. 5,567,811-   U.S. Pat. No. 5,574,146-   U.S. Pat. No. 5,576,427-   U.S. Pat. No. 5,583,013-   U.S. Pat. No. 5,591,722-   U.S. Pat. No. 5,597,909-   U.S. Pat. No. 5,602,244-   U.S. Pat. No. 5,610,300-   U.S. Pat. No. 5,627,053-   U.S. Pat. No. 5,639,873-   U.S. Pat. No. 5,641,515-   U.S. Pat. No. 5,645,897-   U.S. Pat. No. 5,646,265-   U.S. Pat. No. 5,658,873-   U.S. Pat. No. 5,670,633-   U.S. Pat. No. 5,700,920-   U.S. Pat. No. 5,705,629-   U.S. Pat. No. 5,736,152-   U.S. Pat. No. 5,739,169-   U.S. Pat. No. 5,760,395-   U.S. Pat. No. 5,792,747-   U.S. Pat. No. 5,801,005-   U.S. Pat. No. 5,824,311-   U.S. Pat. No. 5,830,880-   U.S. Pat. No. 5,846,225-   U.S. Pat. No. 5,846,945-   U.S. patent Ser. No. 10/667,126-   U.S. patent Ser. No. 11/141,707-   U.S. patent Ser. No. 11/273,640-   U.S. patent Ser. No. 12/134,932-   U.S. Patent Publn. 2005/0261218-   U.S. Patent Publn. 2008/0050744-   Austin-Ward and Villaseca, Revista Medica de Chile, 126(7):838-845,    1998.-   Bagga et al., Cell, 122(4):553-563, 2005.-   Bukowski et al., Clinical Cancer Res., 4(10):2337-2347, 1998.-   Calin and Croce, Nat Rev Cancer, 6(11):857-866, 2006.-   Christodoulides et al., Microbiology, 144(Pt 11):3027-3037, 1998.-   Davidson et al., J. Immunother., 21(5):389-398, 1998.-   Denli et al., Trends Biochem. Sci., 28:196, 2003.-   Diliman, Cancer Biother. Radiopharm., 14(1):5-10, 1999.-   EP 266,032-   Esquela-Kerscher and Slack, Nat Rev Cancer, 6(4):259-269, 2006.-   Froehler et al., Nucleic Acids Res., 14(13):5399-5407, 1986.-   Griffiths-Jones et al., Nucleic Acids Res., 34:D140-D144, 2006.-   Hanibuchi et al., Int. J. Cancer, 78(4):480-485, 1998.-   Hellstrand et al., Acta Oncol., 37(4):347-353, 1998.-   Hui and Hashimoto, Infection Immun., 66(11):5329-5336, 1998.-   Hu-Lieskovan et al., Cancer Res., 65(19):8984-92, 2005.-   Itakura and Riggs, Science, 209:1401-1405, 1980.-   Jeffs et al., Pharm Res., 22(3):362-72, 2005.-   Ju et al., Gene Ther., 7(19):1672-1679, 2000.-   Lagos-Quintana et al., Rna, 9(2):175-179, 2003.-   Lau et al., Science, 294(5543):858-862, 2001.-   Lee and Ambros, Science, 294(5543):862-864, 2001.-   Lee, EMBO J., 21(17):4663-4670, 2002.-   Lim et al., Nature, 433(7027):769-773, 2005.-   Martin et al., Helv. Chim. Acta, 78:486-504, 1995.-   Olsen et al., Dev. Biol., 216:671, 1999.-   PCT Appln. PCT/US89/02323,-   Pietras et al., Oncogene, 17(17):2235-2249, 1998.-   Qin et al., Proc. Natl. Acad. Sci. USA, 95(24):14411-14416, 1998.-   Remington's Pharmaceutical Sciences, 15^(th) Ed., 1035-1038 and    1570-1580, 1990.-   Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3^(rd)    Ed., Cold Spring Harbor Laboratory Press, 2001.-   Sambrook et al., In: DNA microaarays: a molecular cloning manual,    Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2003.-   Sambrook et al., In: Molecular cloning: a laboratory manual, 2^(nd)    Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,    1989.-   Seggerson et al., Dev. Biol., 243:215, 2002.-   Wiemer, Eur J Cancer, 43(10):1529-1544, 2007.

What is claimed is:
 1. A double-stranded, blunt-ended RNA molecule 22 or23 basepairs in length comprising: a) an active strand comprising SEQ IDNO:1 from 5′ to 3′ and b) a fully complementary passenger strandcomprising i) modified nucleotides in the first and last two nucleotidesof the passenger strand; and ii) a terminal modification of thenucleotide at the 5′ end.
 2. The RNA molecule of claim 1, wherein themolecule is 23 basepairs in length and the active strand comprises SEQID NO:2.
 3. The RNA molecule of claim 1, wherein the passenger strandcomprises a modified nucleotide at positions 1, 2, 3, 4, 5, 6, 9, 10,11, 12, 13, 14, 17, 18, 19, 20, 21, 22, and/or 23 relative to SEQ IDNO:4, wherein fewer than half of the total number of nucleotides in thepassenger strand are modified nucleotides.
 4. The RNA molecule of any ofclaims 1-3, wherein the passenger strand comprises modified nucleotideslocated at positions i) 1 and 2 and/or ii) 22 and 23 relative to SEQ IDNO:4.
 5. The RNA molecule of any of claims 2-3, wherein the passengerstrand comprises modified nucleotides located at positions i) 1 and 2and ii) 22 and 23 relative to SEQ ID NO:4.
 6. The RNA molecule of any ofclaims 1-5, wherein the passenger strand comprises a modified nucleotidelocated at positions i) 3 and 4 and/or ii) 19 and 20 relative to SEQ IDNO:4.
 7. The RNA molecule of claim 6, wherein the passenger strandcomprises a modified nucleotide located at positions i) 3 and 4 or ii)19 and 20 relative to SEQ ID NO:4, but not at both positions i) and ii).8. The RNA molecule of any of claims 4-7, wherein the passenger strandcomprises modified nucleotides at positions 5 and 6 relative to SEQ IDNO:4.
 9. The RNA molecule of any of claims 4-7, wherein the passengerstrand comprises a modified nucleotide at positions 9 and 10 relative toSEQ ID NO:4.
 10. The RNA molecule of any of claims 4-7, wherein thepassenger strand comprises a modified nucleotide at positions 11 and 12relative to SEQ ID NO:4.
 11. The RNA molecule of any of claims 4-7,wherein the passenger strand comprises a modified nucleotide atpositions 13 and 14 relative to SEQ ID NO:4.
 12. The RNA molecule of anyof claims 4-7, wherein the passenger strand comprises a modifiednucleotide at positions 17 and 18 relative to SEQ ID NO:4.
 13. The RNAmolecule of claim 4, wherein the number of modified nucleotides in thepassenger strand is four to eight.
 14. The RNA molecule of claim 13,wherein the number of modified nucleotides in the passenger strand isfive to seven.
 15. The RNA molecule of claim 14, wherein the number ofmodified nucleotides in the passenger strand is six.
 16. The RNAmolecule of any of claims 1-15, wherein the passenger strand does nothave a modified nucleotide located at positions 7, 8, 9, 15, and 16relative to SEQ ID NO:4.
 17. The RNA molecule of any of claims 1-16,wherein the passenger strand comprises a modified nucleotide atpositions 1, 2, 21, 22, and 23 relative to SEQ ID NO:4.
 18. The RNAmolecule of any of claims 1-16, wherein the passenger strand comprises amodified nucleotide at positions 1, 2, 3, 4, 22, and 23 relative to SEQID NO:4.
 19. The RNA molecule of any of claims 1-16, wherein thepassenger strand comprises a modified nucleotide at positions 1, 2, 19,20, 22, and 23 relative to SEQ ID NO:4.
 20. The RNA molecule of any ofclaims 1-19, wherein the active strand comprises at least two modifiednucleotides, wherein the modified nucleotides are not in the first twopositions at the 5′ end.
 21. The RNA molecule of any of claims 1-19,wherein the active strand does not have a modified nucleotide in thefirst two positions at the 5′ end.
 22. The RNA molecule of claim 1,wherein the active strand does not comprise a modified nucleotide in thefirst or last two positions from the ends.
 23. The RNA molecule of anyof claims 20-22, wherein the active strand has modified nucleotides atpositions 3, 4, 11, 12, 13, 14, 15, 16, 17, and/or 18 relative to SEQ IDNO:2.
 24. The RNA molecule of claim 23, wherein the active strandcomprises modified nucleotides at positions 15 and 16 relative to SEQ IDNO:2.
 25. The RNA molecule of claim 23, wherein the active strandcomprises modified nucleotides at positions 3 and 4 relative to SEQ IDNO:2.
 26. The RNA molecule of claim 23, wherein the active strandcomprises modified nucleotides at positions 11 and 12 relative to SEQ IDNO:2.
 27. The RNA molecule of claim 23, wherein the active strandcomprises modified nucleotides at positions 11, 12, 13, and 14 relativeto SEQ ID NO:2.
 28. The RNA molecule of claim 23, wherein the activestrand comprises modified nucleotides at positions 17 and 18 relative toSEQ ID NO:2.
 29. The RNA molecule of any of claims 23-28, wherein theactive strand comprises modified nucleotides at positions 11, 12, 15,and 16 relative to SEQ ID NO:2.
 30. The RNA molecule of claim 29,wherein the active strand further comprises modified nucleotides atpositions 13, 14, 17, and 18 relative to SEQ ID NO:2.
 31. The RNAmolecule of any of claims 23-28, wherein the active strand comprisesmodified nucleotides at positions 3, 4, 15, and 16 relative to SEQ IDNO:2.
 32. The RNA molecule of any of claims 1-19, wherein the activestrand does not have any modified nucleotides.
 33. The RNA molecule ofclaim 29, wherein the passenger strand does not comprise any modifiednucleotides except a 5′ terminal modification.
 34. The RNA molecule ofclaim 29, wherein the passenger strand comprises SEQ ID NO:4 andmodified nucleotides at positions 1, 2, 20, 21, 22, and 23 relative toSEQ ID NO:4.
 35. The RNA molecule of claim 29, wherein the passengerstrand comprises SEQ ID NO:4 and modified nucleotides at positions 1, 2,3, 4, 22, and 23 relative to SEQ ID NO:4.
 36. The RNA molecule of any ofclaim 29, wherein the passenger strand comprises SEQ ID NO:4 andmodified nucleotides at positions 1, 2, 19, 20, 22, and 23 relative toSEQ ID NO:4.
 37. The RNA molecule of claim 30, wherein the passengerstrand does not comprise any modified nucleotides except a 5′ terminalmodification.
 38. The RNA molecule of claim 30, wherein the passengerstrand comprises SEQ ID NO:4 and modified nucleotides at positions 1, 2,20, 21, 22, and 23 relative to SEQ ID NO:4.
 39. The RNA molecule ofclaim 30, wherein the passenger strand comprises SEQ ID NO:4 andmodified nucleotides at positions 1, 2, 3, 4, 22, and 23 relative to SEQID NO:4.
 40. The RNA molecule of any of claim 30, wherein the passengerstrand comprises SEQ ID NO:4 and modified nucleotides at positions 1, 2,19, 20, 22, and 23 relative to SEQ ID NO:4. [A-ID 206/P-ID 215]
 41. TheRNA molecule of claim 31, wherein the passenger strand does not compriseany modified nucleotides except a 5′ terminal modification.
 42. The RNAmolecule of claim 31, wherein the passenger strand comprises SEQ ID NO:4and modified nucleotides at positions 1, 2, 21, 22, and 23 relative toSEQ ID NO:4.
 43. The RNA molecule of claim 31, wherein the passengerstrand comprises SEQ ID NO:4 and modified nucleotides at positions 1, 2,3, 4, 22, and 23 relative to SEQ ID NO:4.
 44. The RNA molecule of any ofclaim 31, wherein the passenger strand comprises SEQ ID NO:4 andmodified nucleotides at positions 1, 2, 19, 20, 22, and 23 relative toSEQ ID NO:4.
 45. The RNA molecule of any of claims 1-44, wherein theterminal modification of the passenger strand comprises a loweralkylamine group.
 46. The RNA molecule of any of claims 1-45, whereinthe modified nucleotides have sugar modifications.
 47. The RNA moleculeof claim 46, wherein the sugar modification is 2′-OMe.
 48. Adouble-stranded RNA molecule of 20-23 basepairs in length, wherein theRNA molecule is blunt-ended at both ends, comprising an active strandhaving the sequence of SEQ ID NO:2 and a separate and fullycomplementary passenger strand with a modified nucleotide at the 5′ end,wherein the active strand comprises at least one modified internalnucleotide and wherein the double-stranded RNA molecule is more stablecompared to a double-stranded, blunt-ended RNA molecule lacking anymodification of an internal nucleotide.
 49. A pharmaceutical compositioncomprising the RNA molecule of any of claims 1-48.
 50. A method forproviding miR-34a activity to a cell comprising administering to thecell the RNA molecule of any of claims 1-48.
 51. A method for decreasingcell proliferation comprising administering to the cell an effectiveamount of the RNA molecule of any of claims 1-48.
 52. A method forinducing apoptosis in a cell comprising administering to the cell aneffective amount of the RNA molecule of any of claims 1-48.
 53. A methodfor treating cancer in a patient comprising administering to the patienta pharmaceutical composition comprising the RNA molecule of any ofclaims 1-48.
 54. The method of claim 54, further comprisingadministering to the patient an additional cancer therapy.
 55. Themethod of claim 53, wherein the patient has been diagnosed with cancer.56. A double-stranded, blunt-ended RNA molecule 23 or 24 basepairs inlength comprising: a) an active strand comprising i) SEQ ID NO:5 from 5′to 3′ and ii) at least one modified internal nucleotide; and, b) a fullycomplementary passenger strand comprising a terminal modification of thenucleotide at the 5′ end.
 57. The RNA molecule of claim 56, wherein themolecule is 23 basepairs in length.
 58. The RNA molecule of claim 56,wherein the passenger strand comprises a modified nucleotide atpositions 1 (G), 2 (C), 3 (A), 21 (C), 22 (C), and/or 23 (U) relative toSEQ ID NO:6.
 59. The RNA molecule of any of claims 56-58, wherein thepassenger strand comprises modified nucleotides located at a subset ofpositions, wherein the subset is i) 1 (A), 2 (A), and 3 (C) and/or ii)22 (C), and 23 (A) relative to SEQ ID NO:6.
 60. The RNA molecule of anyof claims 56-59, wherein the passenger strand comprises a modifiednucleotide located at positions i) 1 (A), 2 (A), and 3 (C) relative toSEQ ID NO:6.
 61. The RNA molecule of any of claims 56-60, wherein thepassenger strand comprises a modified nucleotide located at positionsii) 22 (C), and 23 (A) relative to SEQ ID NO:6.
 62. The RNA molecule ofclaim 59, wherein the passenger strand comprises a modified nucleotidelocated at positions 1 (A), 2 (A), 3 (C), 22 (C), and 23 (A) relative toSEQ ID NO:6.
 63. The RNA molecule of claim 59, wherein the passengerstrand comprises a modified nucleotide located at positions i) or ii)relative to SEQ ID NO:6, but not at both positions i) and ii).
 64. TheRNA molecule of any of claims 56-63, wherein the passenger strandfurther comprises a modified nucleotide located at positions 4 (A), 7(A), 8 (G), 9 (C), 10 (U), 11 (A), 12 (A), 13 (C), 14 (U), 19 (U),and/or 21 (C) relative to SEQ ID NO:6.
 65. The RNA molecule of any ofclaim 56, wherein the passenger strand does not have a modifiednucleotide located at positions 5 (U), 6 (C), 15 (A), and/or 16 (C)relative to SEQ ID NO:6.
 66. The RNA molecule of any of claim 56,wherein the number of modified nucleotides in the passenger strand istwo to eight.
 67. The RNA molecule of claim 66, wherein the number ofmodified nucleotides in the passenger strand is three to five.
 68. TheRNA molecule of any of claim 56, wherein the passenger strand comprisesmodified nucleotides at positions 1 (A), 2 (A), 3 (C), 17 (A), 18 (C),21 (C), 22 (C), and 23 (A) relative to SEQ ID NO:6.
 69. The RNA moleculeof any of claim 56, wherein the passenger strand comprises modifiednucleotides at positions 1 (A), 2 (A), 3 (C), 11 (A), 12 (A), 13 (C), 14(U), 21 (C), 22 (C), and 23 (A) relative to SEQ ID NO:6.
 70. The RNAmolecule of any of claim 56, wherein the passenger strand comprisesmodified nucleotides at positions 1 (A), 2 (A), 3 (C), 11 (A), 12 (A),13 (C), 14 (U), 17 (A), 18 (C), 21 (C), 22 (C), and 23 (A) relative toSEQ ID NO:6.
 71. The RNA molecule of any of claims 56-70, wherein theactive strand comprises at least two modified nucleotides, wherein themodified nucleotides are not in the first two positions at the 5′ end.72. The RNA molecule of any of claims 56-70, wherein the active stranddoes not have a modified nucleotide in the first two positions at the 5′end.
 73. The RNA molecule of claim 56, wherein the active strand doesnot comprise a modified nucleotide in the first or last two positionsfrom the ends.
 74. The RNA molecule of any of claims 71-73, wherein theactive strand comprises modified nucleotides at positions 3 (G), 4 (C),11 (G), 12 (U), 13 (A), 14 (G), 15 (C), 16 (C), 17 (U), and/or 18 (G)relative to SEQ ID NO:5.
 75. The RNA molecule of claim 74, wherein theactive strand comprises a modified nucleotide at positions 11 (G) and 12(U) relative to SEQ ID NO:5.
 76. The RNA molecule of claim 74, whereinthe active strand comprises a modified nucleotide at positions 17 (U)and 18 (G) relative to SEQ ID NO:5.
 77. The RNA molecule of claim 74,wherein the active strand comprises a modified nucleotide at positions11 (G), 12 (U), 17 (U), and 18 (G) relative to SEQ ID NO:5.
 78. The RNAmolecule of claim 77, wherein the active strand further comprises amodified nucleotide at positions 3 (G), 4 (C), 13 (A), 14 (G), 15 (C),and/or 16 (C) relative to SEQ ID NO:5.
 79. The RNA molecule of any ofclaims 74-78, wherein the passenger strand does not comprise anymodified nucleotides.
 80. The RNA molecule of claim 68, wherein theactive strand comprises a modified nucleotide at positions 11 (G), 12(U), 17 (U), and 18 (G) relative to SEQ ID NO:5.
 81. The RNA molecule ofclaim 69, wherein the active strand comprises a modified nucleotide atpositions 11 (G), 12 (U), 17 (U), and 18 (G) relative to SEQ ID NO:5.82. The RNA molecule of any of claim 70, wherein the active strandcomprises a modified nucleotide at positions 11 (G), 12 (U), 17 (U), and18 (G) relative to SEQ ID NO:5.
 83. The RNA molecule of any of claims56-82, wherein the terminal modification of the passenger strandcomprises a lower alkylamine group.
 84. The RNA molecule of any ofclaims 56-83, wherein the modified nucleotides have modified sugars. 85.The RNA molecule of claim 84, wherein the sugar modification is 2′-OMe.86. A double-stranded, blunt-ended RNA molecule 23 or 24 basepairs inlength comprising: a) an active strand comprising i) SEQ ID NO:5 from 5′to 3′ and ii) at least one modified internal nucleotide; and, b) a fullycomplementary passenger strand comprising a terminal modification of thenucleotide at the 5′ end and at least one sugar modification atpositions 1 (G), 2 (C), 3 (A), 21 (C), 22 (C) and/or 23 (U) relative toSEQ ID NO:6.
 87. A double-stranded, blunt-ended RNA molecule 23 or 24basepairs in length comprising: a) an active strand comprising at leastbases 1-23 of SEQ ID NO:5 from 5′ to 3′; and, b) a fully complementarypassenger strand comprising: i) a terminal modification of thenucleotide at the 5′ end; ii) a sugar modification in the first threeand the last three nucleotides; and, iii) a sugar modification of thenucleotides at one or more of positions 11 (A), 12 (A), 13 (C), 14 (U),17 (A), and/or 18 (C) relative to SEQ ID NO:6.
 88. A pharmaceuticalcomposition comprising the RNA molecule of any of claims 56-87.
 89. Amethod for providing miR-34c activity to a cell comprising administeringto the cell the RNA molecule of any of claims 56-86.
 90. A method fordecreasing cell proliferation comprising administering to the cell aneffective amount of the RNA molecule of any of claims 56-86.
 91. Amethod for inducing apoptosis in a cell comprising administering to thecell an effective amount of the RNA molecule of any of claims 56-86. 92.A method for treating cancer in a patient comprising administering tothe patient a pharmaceutical composition comprising the RNA molecule ofany of claims 56-86.
 93. The method of claim 92, further comprisingadministering to the patient an additional cancer therapy.
 94. Themethod of claim 92, wherein the patient has been diagnosed with cancer.95. A double-stranded, blunt-ended RNA molecule 22 or 23 basepairs inlength comprising: a) an active strand comprising i) SEQ ID NO:7 from 5′to 3′ and ii) at least one modified internal nucleotide; and, b) a fullycomplementary passenger strand comprising a terminal modification of thenucleotide at the 5′ end.
 96. The RNA molecule of claim 95, wherein themolecule is 23 basepairs in length.
 97. The RNA molecule of claims95-96, wherein the active strand comprises a modified nucleotide atposition 3, 4, 11, 12, 13, 14, 15, 16, 17, and/or 18 in the activestrand.
 98. The RNA molecule of any of claims 95-97, wherein the activestrand comprises at least two modified internal nucleotides.
 99. The RNAmolecule of claim 98, wherein the active strand comprises no more than11 modified internal nucleotides.
 100. The RNA molecule of any of claims95-99, wherein the passenger strand comprises at least one modifiedinternal nucleotide.
 101. The RNA molecule of claim 100, wherein thepassenger strand comprises a modified nucleotide at position 1, 2, 3, 4,9, 10, 11, 12, 13, 14, 19, 21, and/or 22 in the passenger strand. 102.The RNA molecule of any of claims 95-101, wherein the passenger stranddoes not have a modified nucleotide at position 15 and/or
 16. 103. TheRNA molecule of any of claims 95-102, wherein the passenger strandcomprises SEQ ID NO:3.
 104. The RNA molecule of claim 103, wherein thepassenger strand comprises a modified nucleotide at position 1, 2, 3, 5,6, 9, 10, 11, 12, 13, 14, 17, 18, 19, 20, 21, and/or 22 in the passengerstrand.
 105. The RNA molecule of any of claims 95-102, wherein theactive strand comprises SEQ ID NO:5.
 106. The RNA molecule of claim 105,wherein the passenger strand comprises a modified nucleotide at position1, 2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 19, 21, 22, and/or 23 in thepassenger strand.
 107. The RNA molecule of any of claims 95-106, whereinthe active strand comprises the sequence of SEQ ID NO:9.
 108. Adouble-stranded, blunt-ended RNA molecule 22 or 23 basepairs in lengthcomprising: a) an active strand comprising i) SEQ ID NO:9 from 5′ to 3′and ii) a modified nucleotide at position 3, 4, 11, 12, 13, 14, 15, 16,17, and/or 18; b) a fully complementary passenger strand comprising aterminal modification of the nucleotide at the 5′ end.